Methylenetetrahydrofolate reductase inhibitors and uses thereof

ABSTRACT

The present invention provides methylenetetrahydrofolate reductase (MTHFR) inhibitors for use in selective inhibition of cancer cell growth in a mammal. These inhibitors can be a small molecule, an antisense oligonucleotide, a ribozyme, a triple helix forming oligonucleotide, an anti-MTHFR antibody or fragment thereof, an MTHFR mutant or a fragment of MTHFR. The present invention further provides nucleic acids encoding an inhibitor of MTHFR, and vectors comprising these nucleic acids. Also encompassed by the present invention are methods of using the MTHFR inhibitors for selective inhibition of cancer cell growth, and pharmaceutical compositions comprising the MTHFR inhibitors.

FIELD OF THE INVENTION

[0001] The present invention pertains to the field of inhibitors ofcancer cell growth and/or metastasis. More particularly the presentinvention pertains to inhibitors of methylenetetrahydrofolate reductaseand use thereof in the treatment of cancer.

BACKGROUND

[0002] Folic acid derivatives are coenzymes for several criticalsingle-carbon transfer reactions, including reactions in thebiosynthesis of purines, thymidylate and methionine.Methylenetetrahydrofolate reductase (MTHFR; EC 1.5.1.20) catalyses theNADPH-linked reduction of 5,10-methylenetetrahydrofolate to5-methyltetrahydrofolate, a co-substrate for methylation of homocysteineto methionine.

[0003] Hereditary deficiency of MTHFR, an autosomal recessive disorder,is the most common inborn error of folic acid metabolism. A block in theproduction of methyltetrahydrofolate leads to elevated homocysteine withlow to normal levels of methionine. Patients with severe deficiencies ofMTHFR (0-20% activity in fibroblasts) can have variable phenotypes.Developmental delay, mental retardation, motor and gait abnormalities,peripheral neuropathy, seizures and psychiatric disturbances have beenreported in this group, although at least one patient with severe MTHFRdeficiency was asymptomatic. Pathologic changes in the severe forminclude the vascular changes that have been found in other conditionswith elevated homocysteine, as well as reduced neurotransmitter andmethionine levels in the CNS. A milder deficiency of MTHFR (35-50%activity) has been described in patients with coronary artery disease(see below). Genetic heterogeneity is likely, considering the diverseclinical features, the variable levels of enzyme activity, and thedifferential heat inactivation profiles of the reductase in patients'cells.

[0004] MTHFR isolated from porcine liver has been purified tohomogeneity and has been found to be a homodimer of 77-kDa subunits.Partial proteolysis of the porcine peptide has revealed two spatiallydistinct domains: an N-terminal domain of 40 kDa and a C-terminal domainof 37 kDa. The latter domain contains the binding site for theallosteric regulator S-adenosylmethionine.

[0005] The cDNA for human MTHFR has been isolated and mapped, andmutations in the gene have been identified in MTHFR-deficientindividuals (Goyette, et al., (1994) Nat. Genet., 7:195-200).International Patent Application No. PCT/IB00/00442 discloses nucleicacid probes for the MTHFR gene, methods of identifying mutations in theMTHFR gene of individuals with MTHFR deficiency and methods of treatmentfor individuals with MTHFR deficiency involving the provision of afunctional MTHFR gene or protein. The application further teaches thatthe MTHFR deficiency may be associated with a disease, disorder ordysfunction including cancers such as neuroblastomas and colorectalcarcinomas.

[0006] PCT/IB00/00442 also postulates about a method for treating apatient having a cancer by inhibiting MTHFR gene expression or byinhibiting the MTHFR protein. However, given the teaching therein, itremains uncertain whether such a method would be effective in thetreatment of cancer especially in view of the demonstrated link betweenMTHFR deficiency and disease. PCT/IB00/00442 does not discuss how thetreatment of a patient having a cancer by inhibiting MTHFR geneexpression or the MTHFR protein could be implemented or what effect suchinhibition may have on cancer cells. In fact, PCT/IB00/00442 appears toteach that reducing MTHFR activity in a subject will have a deleteriouseffect.

[0007] There remains, therefore, a need for a method of selectivelytargeting cancer cells. In particular for a method that providesspecific inhibition of the growth of cancer cells.

[0008] This background information is provided for the purpose of makingknown information believed by the applicant to be of possible relevanceto the present invention. No admission is necessarily intended, norshould be construed, that any of the preceding information constitutesprior art against the present invention. Publications referred tothroughout the specification are hereby incorporated by reference intheir entireties in this application.

SUMMARY OF THE INVENTION

[0009] An object of the present invention is to providemethylenetetrahydrofolate reductase inhibitors and use thereof. Inaccordance with an aspect of the present invention, there is provided aninhibitor of methylenetetrahydrofolate reductase (MTHFR) for use inselective inhibition of cancer cell growth in a mammal in need of suchtherapy, wherein the inhibitor reduces MTHFR gene expression or MTHFRactivity.

[0010] In accordance with another aspect of the invention, there isprovided a nucleic acid comprising a sequence encoding an inhibitor ofmethylenetetrahydrofolate reductase (MTHFR) for use in selectiveinhibition of cancer cell growth in a mammal in need of such therapy,wherein the inhibitor reduces MTHFR gene expression or MTHFR activityand wherein the inhibitor is an antisense oligonucleotide, a ribozyme, atriple helix forming oligonucleotide, an anti-MTHFR antibody or fragmentthereof, an MTHFR mutant or a fragment of MTHFR. In accordance with yetanother aspect of the invention there is provided a vector comprisingthis nucleic acid.

[0011] In accordance with another aspect of the invention, there isprovided a pharmaceutical composition comprising an inhibitor ofmethylenetetrahydrofolate reductase (MTHFR) for use in selectiveinhibition of cancer cell growth in a mammal in need of such therapy,wherein the inhibitor reduces MTHFR gene expression or MTHFR activityand a pharmaceutically acceptable carrier, diluent or excipient.

[0012] In accordance with another aspect of the invention, there isprovided a use of an MTHFR inhibitor for the manufacture of amedicament, wherein the inhibitor selectively inhibits cancer cellgrowth and wherein the inhibitor reduces MTHFR gene expression or MTHFRactivity.

[0013] In accordance with another aspect of the invention, there isprovided a use of a vector comprising a nucleic acid comprising asequence encoding an inhibitor of methylenetetrahydrofolate reductase(MTHFR) for use in selective inhibition of cancer cell growth in amammal in need of such therapy, wherein the inhibitor reduces MTHFR geneexpression or MTHFR activity and wherein the inhibitor is an antisenseoligonucleotide, a ribozyme, a triple helix forming oligonucleotide, ananti-MTHFR antibody or fragment thereof, an MTHFR mutant or a fragmentof MTHFR, for the manufacture of a medicament, wherein the inhibitorselectively inhibits cancer cell growth and wherein the inhibitorreduces MTHFR gene expression or MTHFR activity.

[0014] In accordance with another aspect of the invention, there isprovided a use of a non-allele specific antisense oligonucleotide atleast 7 nucleotides in length that comprises a sequence that iscomplementary to a gene encoding human MTHFR, wherein theoligonucleotide inhibits human MTHFR gene expression. In accordance withyet another aspect of the invention, there is provided a vectorcomprising a sequence encoding this antisense oligonucleotide.

[0015] In accordance with another aspect of the invention, there isprovided a method of treating stabilizing or preventing cancer in amammal comprising the step of selectively inhibiting cancer cell growthin the mammal by administering an inhibitor of MTHFR, wherein theinhibitor reduces MTHFR gene expression or MTHFR activity.

[0016] In accordance with another aspect of the invention, there isprovided a method of inhibiting growth of cancer cells comprising thestep of contacting said cancer cells with an inhibitor of MTHFR, whereinthe inhibitor does not significantly affect non-cancerous cell growthand wherein the inhibitor reduces MTHFR gene expression or MTHFRactivity.

[0017] In accordance with another aspect of the invention, there isprovided a kit for the use of an MTHFR inhibitor for the manufacture ofa medicament or the methods of the present invention, comprising aninhibitor of MTHFR which selectively inhibits cancer cell growth,wherein the inhibitor reduces MTHFR gene expression or MTHFR activity.

BRIEF DESCRIPTION OF THE FIGURES

[0018]FIG. 1 depicts the total available sequence (SEQ ID NO:1 and NO:2)of human MTHFR cDNA.

[0019]FIG. 2 depicts human MTHFR exons and flanking intronic sequences.The exonic sequences (SEQ ID NOs:3-13) of the human gene are given,along with their sizes and flanking intronic sequences. The base pairlocation of the exons within the cDNA, given in parenthesis, relates tothe published human cDNA base pair numbering (Goyette et al., 1994).Base 1 is 12 bp upstream from the ATG in original cDNA, the equivalentbase is indicated here by an asterisk. Exon 1 contains the ATG startsite (underlined), and exon 11 contains the termination codon(underlined).

[0020]FIG. 3 demonstrates the percent survival of SW620 colon carcinomacells following three rounds of treatment for five hours each round withvarying concentrations of an antisense phosphorothioate oligonucleotideto exon 5 of MTHFR. The cells were allowed to recover after the finaltreatment for a period of two days before the cells were counted. Thevalues are expressed as the percent of cells surviving compared to thenumber of cells which survived after treatment with a controloligonucleotide CTSEX5 (phosphorothioate 5′-GTGACGTAGGACAGCGATGG-3′; SEQID NO:17).

[0021]FIG. 4 demonstrates the percent survival of LOVO colon carcinomacells treated with an MTHFR antisense oligonucleotide after a recoveryperiod of three days, as described for FIG. 3.

[0022]FIG. 5 demonstrates the percent survival of BEC 2 neuroblastomacells treated with varying concentrations of an MTHFR antisenseoligonucleotide and allowed to recover for two days, as described forFIG. 3.

[0023]FIG. 6 demonstrates the percent survival of SK-N-F1 neuroblastomacells treated with varying concentrations of an MTHFR antisenseoligonucleotide after a recovery period of four days, as described forFIG. 3.

[0024]FIG. 7 demonstrates the percent survival of MCF7 breast cancercells that were treated with different concentrations of an MTHFRantisense oligonucleotide and allowed to recover for 2.5 days, asdescribed for FIG. 3.

[0025]FIG. 8 demonstrates the percent survival of SKBr3 breast cancercells treated with varying concentrations of an MTHFR antisenseoligonucleotide after a recovery period of 2.5 days, as described forFIG. 3.

[0026]FIG. 9 demonstrates the percent survival of U87-lacZ glioma cellstreated with varying concentrations of an MTHFR antisenseoligonucleotide after a recovery period of two days, as described forFIG. 3. For this experiment an oligonucleotide with six base pairmismatches CT677 (phosphorothioate 5′-TGCTGTCGGAGCGATAGGTC-3′; SEQ IDNO:18) was used as the control oligonucleotide.

[0027]FIG. 10 demonstrates the percent survival of WG1554 fibroblastcells homozygous for a nonsense mutation in MTHFR which were treatedwith 400 nM MTHFR antisense or control oligonucleotide and allowed torecover for three days, as described for FIG. 3.

[0028]FIG. 11 depicts the growth of fibroblast cell lines in deficientmedia. Two wild type fibroblast cell lines (MCH 51, MCH 75) and an MTHFRnull mutant (WG 1554) were grown in MEM (▪), M− (×), and M−H+ (μ) for 12days. The number of cells for each line was counted using the SRB assayat 3 time points. Each point represents the mean of 3 replicates ±SD.

[0029]FIG. 12 depicts the growth of colon carcinoma cell lines indeficient media. Four colon carcinoma cell lines were grown in MEM (▪),M− (×), and M−H+ (μ) for 12 days. The MTHFR genotype of each coloncarcinoma cell line is indicated in parentheses. The number of cells foreach line was counted using the SRB assay at 3 time points. Each pointrepresents the mean of 3 replicates ±SD.

[0030]FIG. 13 depicts cell survival and MTHFR protein levels aftertreatment with the antisense oligonucleotide EX5. (A) Cells were treatedon three successive days with increasing concentration of EX5 (o). Cellswere also treated with a control oligonucleotide, CTSEX5. The number ofsurviving cells was determined by SRB staining. Cell survival aftertransfection with EX5 is expressed as a % of survival after transfectionwith the control CTSEX5 oligonucleotide. Error bars represent ±SE of themean of 3 experiments, each performed in triplicate. (B) MTHFR proteinlevels after three rounds of treatments with Lipofectin only (mocktransfection), 400 nM of CTSEX5, 200 nM of EX5 or 400 nM of EX5. Cellswere harvested after the third treatment and subjected to Western blotanalysis. The position of the MTHFR protein and the molecular weightmarkers are indicated. Protein levels of β-actin were also assayed byWestern blotting to verify equal loading of samples.

[0031]FIG. 14 depicts a comparison of the cell survival of normal humanfibroblasts, breast carcinoma cells and neuroblastoma lines aftertreatment with 400 nM of EX5. Cells were treated three successive dayswith 400 nM of EX5 and 400 nM of CTSEX5. The number of surviving cellswas determined by SRB staining as described in Materials and Methods.For each cell line, cell survival after transfection with EX5 isexpressed as a % of survival after transfection with the control CTSEX5oligonucleotide. Each value on the graph represents the mean of threereplicates ±SD.

DETAILED DESCRIPTION OF THE INVENTION

[0032] The present invention provides a method of selectively inhibitingthe growth of cancer cells by downregulating MTHFR activity. In thecontext of the present invention, downregulation of MTHFR activity canbe achieved by direct inhibition of the MTHFR protein, or by inhibitionof MTHFR gene expression.

[0033] In view of the prior art regarding the deleterious effects ofMTHFR deficiency, a downregulation of levels of MTHFR protein would beexpected to produce only negative effects. Therefore, a particularlyunexpected result of downregulation of MTHFR protein levels in a mousecancer model was its effectiveness in reducing the number and size oftumours. As described herein, this unexpected finding was furtherdemonstrated following inhibition of MTHFR gene expression usingnon-allele specific antisense oligonucleotides in the treatment ofcancer cells and of normal cells.

[0034] Definitions

[0035] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains.

[0036] The term “non allele-specific” as used herein refers to acompound capable of binding to at least two different MTHFR alleles.

[0037] The term “specifically hybridize” as used herein refers to theability of a nucleic acid to bind detectably and specifically to asecond nucleic acid. Polynucleotides, oligonucleotides and fragmentsthereof specifically hybridize to target nucleic acid strands underhybridization and wash conditions that minimize appreciable amounts ofdetectable binding to nonspecific nucleic acids. High stringencyconditions can be used to achieve specific hybridization as known in theart (for example, see Ausubel, et al., (2000) Current Protocols inMolecular Biology, Wiley & Sons, New york, N.Y.).

[0038] Typically, hybridization and washing conditions are performed athigh stringency according to conventional hybridization procedures.Washing conditions are typically 1-3× SSC, 0.1-1% SDS, 50-70^(E)C, witha change of wash solution after about 5-30 minutes.

[0039] The term “corresponds to” as used herein with reference tonucleic acid sequences means a polynucleotide sequence that is identicalto all or a portion of a reference polynucleotide sequence. Incontradistinction, the term “complementary to” is used herein to meanthat the polynucleotide sequence is identical to all or a portion of thecomplement of a reference polynucleotide sequence. For illustration, thenucleotide sequence “TATAC” corresponds to a reference sequence “TATAC”and is complementary to a reference sequence “GTATA.”

[0040] The following terms are used herein to describe the sequencerelationships between two or more polynucleotides: “reference sequence,”“comparison window,” “sequence identity,” “percentage of sequenceidentity,” and “substantial identity.” A “reference sequence” is adefined sequence used as a basis for a sequence comparison; a referencesequence may be a subset of a larger sequence, for example, as a segmentof a full-length cDNA or gene sequence, or may comprise a complete cDNAor gene sequence. Generally, a reference sequence is at least 20nucleotides in length, frequently at least 25 nucleotides in length, andoften at least 50 nucleotides in length. Since two polynucleotides mayeach (1) comprise a sequence (i.e. a portion of the completepolynucleotide sequence) that is similar between the twopolynucleotides, and (2) may further comprise a sequence that isdivergent between the two polynucleotides, sequence comparisons betweentwo (or more) polynucleotides are typically performed by comparingsequences of the two polynucleotides over a “comparison window” toidentify and compare local regions of sequence similarity.

[0041] A “comparison window”, as used herein, refers to a conceptualsegment of at least 20 contiguous nucleotide positions wherein apolynucleotide sequence may be compared to a reference sequence of atleast 20 contiguous nucleotides and wherein the portion of thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e. gaps) of 20 percent or less as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. Optimal alignment of sequencesfor aligning a comparison window may be conducted by the local homologyalgorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482, by thehomology alignment algorithm of Needleman and Wunsch (1970) J. Mol.Biol. 48:443, by the search for similarity method of Pearson and Lipman(1988) Proc. Natl. Acad. Sci. (U.S.A.) 85:2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package Release 7.0, Genetics ComputerGroup, 573 Science Dr., Madison, Wis.), or by inspection, and the bestalignment (i.e. resulting in the highest percentage of homology over thecomparison window) generated by the various methods is selected.

[0042] The term “sequence identity” means that two polynucleotidesequences are identical (i.e. on a nucleotide-by-nucleotide basis) overthe window of comparison. The term “percentage of sequence identity” iscalculated by comparing two optimally aligned sequences over the windowof comparison, determining the number of positions at which theidentical nucleic acid base (e.g. A, T, C, G, U, or I) occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison (i.e. the window size), and multiplying the result by 100 toyield the percentage of sequence identity.

[0043] The term “substantial identity” as used herein denotes acharacteristic of a polynucleotide sequence, wherein the polynucleotidecomprises a sequence that has at least 30 percent sequence identity,often at least 50 percent sequence identity, and more usually at least60 percent sequence identity as compared to a reference sequence over acomparison window of at least 20 nucleotide positions, frequently over awindow of at least 25-50 nucleotides, wherein the percentage of sequenceidentity is calculated by comparing the reference sequence to thepolynucleotide sequence which may include deletions or additions whichtotal 20 percent or less of the reference sequence over the window ofcomparison.

[0044] MTHFR Inhibitors

[0045] The present invention provides compounds that selectively inhibitthe growth of cancer cells by downregulating MTHFR activity in a mammal(e.g., a human). The extent of selective inhibition is generallysufficient to treat, stabilize, or prevent cancer in the mammal. In thecontext of the present invention, selective inhibition means that thegrowth of cancer cells is inhibited substantially more than the growthof normal cells. In a specific embodiment of the present inventioncancer cell growth is inhibited by an MTHFR inhibitor under conditionsin which the growth of normal cells also treated with the MTHFRinhibitor is fully or partially unaffected. When the growth of normalcells is partially affected by contact with the MTHFR inhibitor, thedifference between the affect on cancer cells and on normal cells issuch that the cancer cells are preferentially inhibited and/or killed bycontact with the MTHFR inhibitor.

[0046] The inhibitors according to the present invention can beantisense oligonucleotides, biologically inactive MTHFR proteins orfragments, peptides, small molecule inhibitors or antibodies.

[0047] (i) Antisense Oligonucleotides

[0048] “Targeting” an antisense compound to a particular nucleic acid,in the context of this invention, is a multistep process. The processusually begins with the identification of a nucleic acid sequence whosefunction is to be modulated. In the present invention, the target is thegene encoding MTHFR. The targeting process also includes determinationof a site or sites within this gene for the antisense interaction tooccur such that the desired effect, i.e. modulation of expression of theprotein encoded by the gene, will result.

[0049] Generally, there are five regions of a gene that may be targetedfor antisense modulation: the 5′ untranslated region (5′-UTR), thetranslation initiation or start codon region, the open reading frame(ORF), the translation termination or stop codon region and the 3′untranslated region (3′-UTR).

[0050] The terms “translation initiation codon” and “start codon” canencompass many codon sequences, even though the initiator amino acid ineach instance is typically methionine in eukaryotes. It is also known inthe art that eukaryotic genes may have two or more alternative startcodons, any one of which may be preferentially utilized for translationinitiation in a particular cell type or tissue, or under a particularset of conditions. In the context of the present invention, “startcodon” and “translation initiation codon” refer to the codon or codonsthat are used in vivo to initiate translation of an mRNA moleculetranscribed from a gene encoding MTHFR regardless of the sequence(s) ofsuch codons.

[0051] As is known in the art, some eukaryotic transcripts are directlytranslated, however, most mammalian ORFs contain one or more sequences,known as “introns,” which are excised from a transcript before it istranslated; the expressed (unexcised) portions of the ORF are referredto as “exons” (Alberts et al., (1983) Molecular Biology of the Cell,Garland Publishing Inc., New York, pp. 411-415). In the context of thepresent invention, both introns and exons may serve as targets forantisense.

[0052] In some instances, an ORF may also contain one or more sites thatmay be targeted for antisense due to some functional significance invivo. Examples of the latter types of sites include intragenic stem-loopstructures (see, for example, U.S. Pat. No. 5,512,438) and, inunprocessed mRNA molecules, intron/exon splice sites. In addition, mRNAmolecules possess a 5′ cap region that may also serve as a target forantisense. The 5′ cap of a mRNA comprises an N⁷-methylated guanosineresidue joined to the 5′-most residue of the mRNA via a 5′-5′triphosphate linkage. The 5′ cap region of a mRNA is considered toinclude the 5′ cap structure itself as well as the first 50 nucleotidesadjacent to the cap.

[0053] In accordance with the present invention, the antisenseoligonucleotides are non allele-specific, therefore, regions of the geneto be targeted are those that are conserved, i.e. show no sequencedifference, among the different alleles of the MTHFR gene. In oneembodiment of the present invention, the antisense oligonucleotides aretargeted to all or part of one of exons 1-11 or other MTHFR exon(Goyette et al., (1998) Mammalian Genome 9:652-656). In a relatedembodiment, the antisense oligonucleotides are targeted to exon 5 of theMTHFR gene. In a further related embodiment of the present invention,the antisense oligonucleotide has the sequence5′-AGCTGCCGAAGGGAGTGGTA-3′ (SEQ ID NO:16) and binds to nucleotides796-815 of exon 5 of the MTHFR gene.

[0054] In accordance with the present invention, the antisenseoligonucleotide binds at least 70% of the human MTHFR alleles. In arelated embodiments the antisense oligonucleotide binds at least 80%, orat least 90% of the human MTHFR alleles. In another embodiment of thepresent invention, the antisense nucleic acid does not bind to a regionof the MTHFR gene that contains a polymorphic site.

[0055] Once the target site or sites have been identified,oligonucleotides are chosen that are sufficiently complementary (i.e.hybridize with sufficient strength and specificity) to the target togive the desired result.

[0056] The antisense oligonucleotides in accordance with the presentinvention are selected from a sequence complementary to the MTHFR genesuch that the sequence exhibits the least likelihood of formingduplexes, hair-pins, or of containing homooligomer/sequence repeats. Theoligonucleotide may further contain a GC clamp. These properties can bedetermined qualitatively using commercially available computer software,for example, the computer modeling program OLIGO® Primer AnalysisSoftware, Version 5.0 (distributed by National Biosciences, Inc.,Plymouth, Minn.).

[0057] In order to be effective, antisense oligonucleotides aretypically between 7 and 350 nucleotides in length. In one embodiment ofthe present invention the antisense oligonucleotides comprise from atleast about 7 to about 50 nucleotides, or nucleotide analogues. Inrelated embodiments the antisense oligonucleotides comprise from about15 to about 25 nucleotides, or nucleotide analogues, or from about 18 toabout 22 nucleotides, or nucleotide analogues.

[0058] It is understood in the art that an antisense oligonucleotideneed not have 100% identity with its target sequence. The presentinvention, therefore, contemplates antisense oligonucleotides that have100% sequence identity with the target sequence as well as those thathave a sequence that is at least about 75% identical to the targetsequence. In one embodiment of the present invention, the antisenseoligonucleotides have a sequence that is at least about 90% identical.In a related embodiment, they have a sequence that is at least about 95%identical with the target sequence, allowing for gaps or mismatches ofseveral bases. In accordance with the present invention, the antisenseoligonucleotide is less than 50% identical to the reverse complement ofa region in another human expressed sequence (EST) or, in other reportedhuman ESTs. Identity can be determined, for example, by using the BLASTNprogram of the University of Wisconsin Computer Group (GCG) software.

[0059] Alternatively an antisense oligonucleotide of the presentinvention can be defined by its ability to specifically hybridize to thetarget MTHFR gene, as determined using standard techniques known toworkers skilled in the art (e.g. hybridization assays).

[0060] In the context of the present invention, the term“oligonucleotide” refers to an antisense oligomer or polymer ofribonucleic acid (RNA), deoxyribonucleic acid (DNA), modified RNA orDNA, or RNA or DNA mimetics. This term, therefore, includesoligonucleotides composed of naturally-occurring nucleobases, sugars andcovalent internucleoside (backbone) linkages as well as oligonucleotideshaving non-naturally-occurring portions which function similarly. Suchmodified or substituted oligonucleotides are often preferred over nativeforms because of desirable properties such as, enhanced cellular uptake,enhanced affinity for nucleic acid target and increased stability in thepresence of nucleases.

[0061] Examples of modified or substituted antisense compounds useful inthis invention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. In accordance with the presentinvention, oligonucleotides having modified backbones include those thatretain a phosphorus atom in the backbone and those that do not have aphosphorus atom in the backbone. For the purposes of this specification,and as sometimes referenced in the art, modified oligonucleotides thatdo not have a phosphorus atom in their internucleoside backbone can alsobe considered to be oligonucleosides.

[0062] Exemplary modified oligonucleotide backbones include, forexample, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates including3′amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acidforms are also included.

[0063] Exemplary modified oligonucleotide backbones that do not includea phosphorus atom are formed by short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatom and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. Such backbones include morpholinolinkages (formed in part from the sugar portion of a nucleoside);siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyland thioformacetyl backbones; methylene formacetyl and thioformacetylbackbones; alkene containing backbones; sulfamate backbones;methyleneimino and methylenehydrazino backbones; sulfonate andsulfonamide backbones; amide backbones; and others having mixed N, O, Sand CH₂ component parts.

[0064] The present invention also contemplates oligonucleotide mimeticsin which both the sugar and the internucleoside linkage of thenucleotide units are replaced with novel groups. The base units aremaintained for hybridization with an appropriate nucleic acid targetcompound. An example of such an oligonucleotide mimetic, which has beenshown to have excellent hybridization properties, is a peptide nucleicacid (PNA) (Nielsen et al., (1991) Science, 254:1497-1500). In PNAcompounds, the sugar-backbone of an oligonucleotide is replaced with anamide containing backbone, in particular an aminoethylglycine backbone.The nucleobases are retained and are bound directly or indirectly toaza-nitrogen atoms of the amide portion of the backbone.

[0065] Modified oligonucleotides may also contain one or moresubstituted sugar moieties. For example, oligonucleotides may comprisesugars with one of the following substituents at the 2′ position: OH; F;O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; orO-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may besubstituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl andalkynyl. Examples of such groups are: O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Alternatively, the oligonucleotides may comprise one of the followingsubstituents at the 2′ position: C₁ to C₁₀ lower alkyl, substitutedlower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN,Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.Specific examples include 2′-methoxyethoxy (2′-O—CH₂ CH₂ OCH₃, alsoknown as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., (1995) Helv.Chim. Acta, 78:486-504), 2′-dimethylaminooxyethoxy (O(CH₂)₂ ON(CH₃)₂group, also known as 2′-DMAOE), 2′-methoxy (2′-O—CH₃), 2′-aminopropoxy(2′-OCH₂ CH₂ CH₂ NH₂) and 2′-fluoro (2′-F).

[0066] Similar modifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide. Oligonucleotides may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar.

[0067] Oligonucleotides may also include modifications or substitutionsto the nucleobase. As used herein, “unmodified” or “natural” nucleobasesinclude the purine bases adenine (A) and guanine (G), and the pyrimidinebases thymine (T), cytosine (C) and uracil (U). Modified nucleobasesinclude other synthetic and natural nucleobases such as 5-methylcytosine(5-me-C), 5- hydroxymethyl cytosine, xanthine, hypoxanthine,2-aminoadenine, 6-methyl and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil andcytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808; TheConcise Encyclopedia Of Polymer Science And Engineering, (1990) pp858-859, Kroschwitz, J. I., ed. John Wiley & Sons; Englisch et al.,(1991) Angewandte Chemie, Int. Ed., 30:613; and Sanghvi, Y. S., (1993)Antisense Research and Applications, pp 289-302, Crooke, S. T. andLebleu, B., ed., CRC Press. Certain of these nucleobases areparticularly useful for increasing the binding affinity of theoligomeric compounds of the invention. These include 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., (1993) AntisenseResearch and Applications, pp 276-278, Crooke, S. T. and Lebleu, B.,ed., CRC Press, Boca Raton).

[0068] Another oligonucleotide modification included in the presentinvention is to chemically link to the oligonucleotide one or moremoieties or conjugates which enhance the activity, cellular distributionor cellular uptake of the oligonucleotide. Such moieties include, butare not limited to, lipid moieties such as a cholesterol moiety(Letsinger et al., (1989) Proc. Natl. Acad. Sci. USA, 86:6553-6556),cholic acid (Manoharan et al., (1994) Bioorg. Med. Chem. Let.,4:1053-1060), a thioether, e.g. hexyl-S-tritylthiol (Manoharan et al.,(1992) Ann. N.Y. Acad. Sci., 660:306-309; Manoharan et al., (1993)Bioorg. Med. Chem. Lett., 3:2765-2770), a thiocholesterol (Oberhauser etal., (1992) Nucl. Acids Res., 20:533-538), an aliphatic chain, e.g.dodecandiol or undecyl residues (Saison-Behmoaras et al., (1991) EMBOJ., 10:1111-1118; Kabanov et al., (1990) FEBS Lett., 259:327-330;Svinarchuk et al., (1993) Biochimie, 75:49-54), a phospholipid, e.g.di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., (1995)Tetrahedron Lett., 36:3651-3654; Shea et al., (1990) Nucl. Acids Res.,18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan etal., (1995) Nucleosides & Nucleotides, 14:969-973), or adamantane aceticacid (Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654), apalmityl moiety (Mishra et al., (1995) Biochim. Biophys. Acta,1264:229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., (1996) J.Pharmacol. Exp. Ther., 277:923-937).

[0069] One skilled in the art will recognise that it is not necessaryfor all positions in a given oligonucleotide to be uniformly modified.The present invention, therefore, contemplates the incorporation of morethan one of the aforementioned modifications into a singleoligonucleotide or even at a single nucleoside within theoligonucleotide. The present invention further includes antisensecompounds that are chimeric compounds. These oligonucleotides typicallycontain at least one region wherein the oligonucleotide is modified soas to confer upon the oligonucleotide increased resistance to nucleasedegradation, increased cellular uptake, and/or increased bindingaffinity for the target nucleic acid. An additional region of theoligonucleotide may serve as a substrate for enzymes capable of cleavingRNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellularendonuclease which cleaves the RNA strand of an RNA:DNA duplex.Activation of RNase H, therefore, results in cleavage of the RNA target,thereby greatly enhancing the efficiency of oligonucleotide inhibitionof gene expression. Consequently, comparable results can often beobtained with shorter oligonucleotides when chimeric oligonucleotidesare used, compared to phosphorothioate deoxyoligonucleotides hybridizingto the same target region. Cleavage of the RNA target can be routinelydetected by gel electrophoresis and, if necessary, associated nucleicacid hybridization techniques known in the art.

[0070] In the context of the present invention, an antisenseoligonucleotide is “nuclease resistant” when it has either been modifiedsuch that it is not susceptible to degradation by DNA and RNA nucleasesor alternatively has been placed in a delivery vehicle which in itselfprotects the oligonucleotide from DNA or RNA nucleases. Nucleaseresistant oligonucleotides include, for example, methyl phosphonates,phosphorothioates, phosphorodithioates, phosphotriesters, and morpholinooligomers. Suitable delivery vehicles for conferring nuclease resistanceinclude, for example, liposomes.

[0071] The present invention further contemplates antisenseoligonucleotides that contain groups for improving the pharmacokineticproperties of the oligonucleotide, or groups for improving thepharmacodynamic properties of the oligonucleotide.

[0072] In one embodiment of the present invention, the antisenseoligonucleotide is a phosphorothioate nucleic acid in which anon-bridging phosphoryl oxygen in one or more of the nucleotides isreplaced with sulphur. In a related embodiment, the antisenseoligonucleotide is the phosphorothioate oligonucleotide with thesequence 5′-AGCTGCCGAAGGGAGTGGTA-3′ (SEQ ID NO:16), which binds to exon5 of the MTHFR gene.

[0073] The antisense oligonucleotides of the present invention can beprepared by conventional techniques well-known to those skilled in theart. For example, the oligonucleotides can be prepared using solid-phasesynthesis using commercially available equipment, such as the equipmentavailable from Applied Biosystems Canada Inc., Mississauga, Canada. Asis well-known in the art, modified oligonucleotides, such asphosphorothioates and alkylated derivatives, can also be readilyprepared by similar methods.

[0074] Alternatively, the antisense oligonucleotides of the presentinvention can be prepared by enzymatic digestion of the naturallyoccurring MTHFR gene by methods known in the art.

[0075] Antisense oligonucleotides can also be prepared by recombinantDNA techniques. The present invention, therefore, encompasses expressionvectors comprising nucleic acid sequences that encode one or moreantisense oligonucleotide that targets the MTHFR gene. The antisenseoligonucleotide(s) encoded by such expression vectors is expressed in asuitable host cell. Suitable expression vectors can be readilyconstructed using procedures known in the art. Examples of suitablevectors include, but are not limited to, plasmids, phagemids, cosmids,bacteriophages, baculoviruses, retroviruses, and RNA and DNA viruses.Generally, the viral vectors are replication deficient by are capable ofexpression f the antisense oligonucleotide(s).

[0076] One skilled in the art will understand that selection of theappropriate host cell for expression of the antisense oligonucleotidewill be dependent upon the vector chosen. Examples of host cellsinclude, but are not limited to, bacterial, yeast, insect, plant andmammalian cells.

[0077] One skilled in the art will also understand that the expressionvector may further include regulatory elements required for efficienttranscription or translation of the antisense oligonucleotide sequences.Examples of regulatory elements that can be incorporated into the vectorinclude, but are not limited to, transcriptional elements such aspromoters, enhancers, terminators, and polyadenylation signals. Thepresent invention, therefore, provides vectors comprising one or moreregulatory elements operatively linked to a nucleic acid sequenceencoding an antisense oligonucleotide. One skilled in the art willappreciate that selection of suitable regulatory elements is dependenton the host cell chosen for expression of the antisense oligonucleotideand that such elements may be derived from a variety of sources,including bacterial, fungal, viral, mammalian or insect genes.

[0078] In the context of the present invention, the expression vectormay additionally contain a reporter gene. Suitable reporter genesinclude, but are not limited to, β-galactosidase, green fluorescentprotein, red fluorescent protein, luciferase, and β-glucuronidase.Incorporation of a reporter gene into the expression vector allowstranscription of the antisense oligonucleotide to be monitored bydetection of a signal generated by expression of the reporter gene.

[0079] In accordance with the present invention, the expression vectorscan be introduced into a suitable host cell or tissue by one of avariety of methods known in the art. These methods include, for example,stable or transient transfection, lipofection, electroporation, andinfection with recombinant viral vectors. Methods of constructingexpression vectors and introducing these vectors into host cells arewell-known in the art, and are generally described in Sambrook et al.,(1992) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press;Ausubel et al., (2000) Current Protocols in Molecular Biology, Wiley &Sons, New York.

[0080] In a related embodiment of the present invention the MTHFRinhibitor is an oligonucleotide that hybridizes to and forms triplehelix structures at the 5′ terminus of the MTHFR gene and can be used toblock transcription. The triple helix forming oligonucleotides can beprepared as described above in relation to the antisenseoligonucleotides. Similarly, nucleic acids encoding the triple helixforming oligonucleotide can be cloned into a vector as described above.

[0081] The ability of the antisense oligonucleotides or triple helixforming oligonucleotides of the present invention to inhibit MTHFR geneexpression can be determined by a number of techniques known to oneskilled in the art. For example, the level of MTHFR mRNA can bedetermined by standard Northern blot analysis, and/or the level of MTHFRprotein can be determined by standard Western blot analysis. Methods ofconducting these methods are well-known to workers skilled in art (see,for example, Ausubel et al., (2000) Current Protocols in MolecularBiology, Wiley & Sons, New York: Coligan, et al., (2001) CurrentProtocols in Protein Science, Wiley & Sons, New York). In one embodimentof the present invention, the level of MTHFR protein is determined bymeasuring the level of MTHFR enzymatic activity, as described inChristensen, et al., (1997) Arterioscler. Thromb. Vasc. Bio.,17:573-596. In an alternate embodiment, the level of MTHFR protein canbe assessed by measuring the resulting increase in cellular levels ofhomocysteine, or decrease in 5-methyltetrahydrofolate or methionine, asdescribed herein.

[0082] (ii) Ribozymes

[0083] In one embodiment of the present invention the MTHFR inhibitor isa ribozyme that specifically targets RNA encoding MTHFR. Ribozymes areRNA molecules having an enzymatic activity which is able to repeatedlycleave other separate RNA molecules in a nucleotide base sequencespecific manner. Such enzymatic RNA molecules can be targeted tovirtually any RNA transcript, and efficient cleavage achieved in vitro.Kim et al., 84 Proc. Nati. Acad. Sci. USA 8788, 1987; Haseloff andGerlach, 334 Nature 585, 1988; Cech, 260 JAMA 3030, 1988; and Jefferieset al., 17 Nucleic Acids Research 1371, 1989.

[0084] Ribozymes act by first binding to a target RNA. Such bindingoccurs through the target RNA binding portion of a ribozyme which isheld in close proximity to an enzymatic portion of the RNA which acts tocleave the target RNA. Thus, the ribozyme first recognizes and thenbinds a target RNA through complementary base-pairing, and once bound tothe correct site, acts enzymatically to cut the target RNA. Strategiccleavage of such a target RNA will destroy its ability to directsynthesis of an encoded protein. After a ribozyme has bound and cleavedits RNA target it is released from that RNA to search for another targetand can repeatedly bind and cleave new targets.

[0085] Hammerhead ribozymes comprise a hybridizing region which iscomplementary in nucleotide sequence to at least part of the target RNA,and a catalytic region which is adapted to cleave the target RNA. Thehybridizing region contains nine (9) or more nucleotides. Therefore, thehammerhead ribozymes of the present invention have a hybridizing regionwhich is complementary to a gene encoding MTHFR and is at least ninenucleotides in length. The construction and production of such ribozymesis well known in the art and is described more fully in Haseloff andGerlach, 1988, Nature, 334:585-591.

[0086] The ribozymes of the present invention also include RNAendoribonucleases (hereinafter “Cech-type ribozymes”) such as the onewhich occurs naturally in Tetrahymena thermophila (known as the IVS, orL-19 IVS RNA) and which has been extensively described by Thomas Cechand collaborators (Zaug, et al., 1984, Science, 224:574-578; Zaug andCech, 1986, Science, 231:470-475; Zaug, et al., 1986, Nature,324:429-433; published International patent application No. WO 88/04300by University Patents Inc.; Been and Cech, 1986, Cell, 47:207-216). TheCech endoribonucleases have an eight base pair active site whichhybridizes to a target RNA sequence whereafter cleavage of the targetRNA takes place.

[0087] There is a narrow range of binding free-energies between aribozyme and its substrate that will produce maximal ribozyme activity.Such binding energy can be optimized by making ribozymes with G to I andU to BrU substitutions (or equivalent substitutions) in thesubstrate-binding arms. This allows manipulation of the bindingfree-energy without actually changing the target recognition sequence,the length of the two substrate-binding arms, or the enzymatic portionof the ribozyme. The shape of the free-energy vs. ribozyme activitycurve can be readily determined using data from experiments in whicheach base (or several bases) is modified or unmodified, and without thecomplication of changing the size of the ribozyme/substrate interaction.

[0088] Such experiments will indicate the most active ribozymestructure. The use of modified bases thus permits “fine tuning” of thebinding free energy to assure maximal ribozyme activity. In addition,replacement of such bases, e.g., I for G, may permit a higher level ofsubstrate specificity when cleavage of non-target RNA is a problem.

[0089] The ability of the ribozymes of the present invention to inhibitMTHFR mRNA expression can be determined by a number of techniques knownto one skilled in the art. For example, the level of MTHFR protein canbe determined by standard Western blot analysis. Techniques ofconducting this method are well-known to workers skilled in art (see,for example, Ausubel et al., (2000) Current Protocols in MolecularBiology, Wiley & Sons, New York: Coligan, et al., (2001) CurrentProtocols in Protein Science, Wiley & Sons, New York). In one embodimentof the present invention, the level of MTHFR protein is determined bymeasuring the level of MTHFR enzymatic activity, as described inChristensen, et al., (1997) Arterioscler. Thromb. Vasc. Bio.,17:573-596. In an alternate embodiment, the level of MTHFR protein canbe assessed by measuring the resulting increase in cellular levels ofhomocysteine, or decrease in 5-methyltetrahydrofolate or methionine, asdescribed herein.

[0090] (iii) Biologically Inactive MTHFR Protein or Fragments of anMTHFR Protein

[0091] The present invention also contemplates the use of a biologicallyinactive MTHFR proteins or fragments of an MTHFR protein that interferewith the action of the wild-type protein and thus, acts as inhibitors ofMTHFR activity.

[0092] Candidate inhibitory fragments can be selected from randomfragments generated from the wild-type MTHFR protein. Methods forgenerating the candidate polypeptide fragments are well known to workersskilled in the art and include, but are not limited to, enzymatic,chemical or mechanical cleavage of the native protein, expression ofnucleic acids encoding such fragments, etc. Biologically inactive MTHFRproteins can be generated by a variety of techniques known to a workerskilled in the art. For example, by site-directed or random mutagenesistechniques of nucleic acids encoding the protein, or by inactivation ofthe protein by chemical or physical means.

[0093] The ability of the biologically inactive MTHFR proteins orfragments to interfere with the wild-type MTHFR activity can bedetermined by standard techniques, for example, using the methoddescribed by Christensen, et al., (1997) Arterioscler. Thromb. Vasc.Bio., 17:573-596, or competitive binding studies.

[0094] (iv) Peptide Inhibitors

[0095] The present invention also provides for polypeptides and peptidesthat bind to and inhibit the activity of the MTHFR protein. Oneexemplary method of identifying such peptides is by phage displaytechniques. Phage display libraries of random short peptides arecommercially available, e.g. from New England Biolabs, Inc., and areutilized through an in vitro selection process known as “panning”. Inits simplest form, panning involves first incubating the library ofphage displayed peptides with a plate, or bead, coated with the targetmolecule, then washing away unbound phage particles, and finally elutingthe specifically bound phage. For the purposes of the present invention,the target molecule is the MTHFR protein, or a fragment thereof.

[0096] The peptide(s) displayed by the specifically-binding phage arethen isolated and sequenced by standard techniques known to thoseskilled in the art. In some instances the binding strength of theisolated peptide is then tested using standard techniques. The abilityof the peptides to inhibit MTHFR activity can also be determined usingassays known in the art and as described herein.

[0097] (v) Small Molecule Inhibitors

[0098] Potential inhibitory compounds are screened from large librariesof synthetic or natural compounds. Numerous means are currently used forrandom and directed synthesis of saccharide, peptide, and nucleic acidbased compounds and are well-known in the art. Synthetic compoundlibraries are commercially available from a number of companiesincluding Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex(Princeton, N.J.), Brandon Associates (Merrimack, N.H.), and Microsource(New Milford, Conn.). A rare chemical library is available from Aldrich(Milwaukee, Wis.). Combinatorial libraries are also available and can beprepared according to standard procedures. Alternatively, libraries ofnatural compounds in the form of bacterial, fungal, plant, and animalextracts are available from, e.g., Pan Laboratories (Bothell, Wash.) orMycoSearch (North Carolina), or are readily producible. Additionally,natural and synthetically produced libraries and compounds are readilymodified through conventional chemical, physical, and biochemical means.

[0099] These libraries can be screened for their ability to inhibit theactivity of the MTHFR protein (for example, using the method describedby Christensen, et al., (1997) Arterioscler. Thromb. Vasc. Bio.,17:573-596) or to inhibit expression of the MTHFR gene by techniquesknown in the art, e.g. nucleic acid binding assays, gel shift assays,and the like.

[0100] (vi) Antibodies

[0101] The present invention also contemplates the use of antibodies,and antibody fragments, raised against the MTHFR protein, or fragmentsthereof, as inhibitors of MTHFR activity.

[0102] For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others can be immunized by injectionwith the MTHFR protein, or with a fragment or oligopeptide thereof thathas immunogenic properties. Depending on the host species, variousadjuvants may be used to increase immunological response. Such adjuvantsinclude, but are not limited to, Freund's adjuvant, mineral gels such asaluminum hydroxide, and surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, Keyhole limpethemolysin (KLH), and dinitrophenol. Examples of adjuvants used in humansinclude, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum.

[0103] The oligopeptides, peptides, or fragments used to induceantibodies to MTHFR can have an amino acid sequence consisting of aslittle as about 5 amino acids. In one embodiment of the presentinvention, amino acid sequences of at least about 10 amino acids areused. These oligopeptides, peptides, or fragments can be identical to aportion of the amino acid sequence of the natural protein that containsthe entire amino acid sequence of a small, naturally occurring molecule.If required, short stretches of MTHFR amino acids can be fused withthose of another protein, such as KLH, and antibodies to the chimericmolecule can be produced.

[0104] Monoclonal antibodies to MTHFR can be prepared using techniquesthat provide for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique (see, for example, Kohler, G. et al. (1975) Nature256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42;Cote, R. J. et al. (1983) Proc. Natl. Acad. Sci. USA, 80:2026-2030; andCole, S. P. et al. (1984) Mol. Cell Biol. 62:109-120). For example, themonoclonal antibodies according to the present invention can be obtainedby immunizing animals, such as mice or rats, with purified MTHFR. Spleencells isolated from the immunized animals are then immortalized usingstandard techniques. Those isolated immortalized cells whose culturesupernatant contains an antibody that causes an inhibition of theactivity of MTHFR with an IC₅₀ of less than 100 ng/ml are then selectedand cloned using techniques that are familiar and known to one skilledin the art. The monoclonal antibodies produced by these clones are thenisolated according to standard protocols.

[0105] The immortalization of the spleen cells of the immunized animalscan be carried out by fusing these cells with a myeloma cell line, suchas P3X63-Ag 8.653 (ATCC CRL 1580) according to the method in (1980) J.Imm. Meth. 39:285-308. Other methods known to a person skilled in theart can also be used to immortalize spleen cells. In order to detectimmortalized cells that produce the desired antibody against the MTHFRprotein, a sample of the culture supernatant is tested using an enzymelinked immunosorbent assay (ELISA) for reactivity with MTHFR. In orderto obtain those antibodies that inhibit the enzymatic activity of MTHFR,the culture supernatant of clones that produce antibodies that bind toMTHFR is additionally examined for inhibition of MTHFR activity using anappropriate assay, such as those described herein. Those clones whoseculture supernatant shows the desired inhibition of MTHFR activity areexpanded and the antibodies produced by these clones are isolatedaccording to known methods.

[0106] In addition, techniques developed for the production of “chimericantibodies,” such as the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity, can be used (Morrison, S. L. et al. (1984)Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984)Nature 312:604-608; and Takeda, S. et al. (1985) Nature 314:452454).Alternatively, techniques described for the production of single chainantibodies can be adapted, using methods known in the art, to produceMTHFR-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, can be generated bychain shuffling from random combinatorial immunoglobulin libraries (see,for example, Burton D. R. (1991) Proc. Natl. Acad. Sci. USA,88:10134-10137).

[0107] Antibodies can also be produced by inducing in vivo production inthe lymphocyte population or by screening immunoglobulin libraries orpanels of highly specific binding reagents as disclosed in theliterature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299).

[0108] Antibody fragments which contain specific binding sites for MTHFRcan also be generated. For example, such fragments include, but are notlimited to, F(ab′)2 fragments produced by pepsin digestion of theantibody molecule and Fab fragments generated by reducing the disulphidebridges of the F(ab′)2 fragments. Alternatively, Fab expressionlibraries can be constructed to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity (see, for example,Huse, W. D. et al. (1989) Scienc,e 246:1275-1281).

[0109] Various immunoassays can be used for screening to identifyantibodies having the desired specificity. Numerous protocols forcompetitive binding or immunoradiometric assays using either polyclonalor monoclonal antibodies with established specificities are well knownin the art. Such immunoassays typically involve the measurement ofcomplex formation between MTHFR and its specific antibody. Examples ofsuch techniques include ELISAs, radioimmunoassays (RIAs), andfluorescence activated cell sorting (FACS). Alternatively, a two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering MTHFR epitopes, or a competitive binding assay canbe used (see, Maddox, D. E. et al. (1983) J. Exp. Med. 158:1211-1216).These and other assays are well known in the art (see, for example,Hampton, R. et al. (1990) Serological Methods: A Laboratory Manual, APSPress, St Paul, Minn., Section IV; Coligan, J. E. et al. (1997, andperiodic supplements) Current Protocols in Immunology, Wiley & Sons, NewYork, N.Y.; Maddox, D. E. et al. (1983) J. Exp. Med. 158:1211-1216).

[0110] Selection of MTHFR Inhibitors

[0111] In order for the inhibitors of the present invention to beeffective, they must reduce the activity of MTHFR in cancer cells to anappropriate extent. As described above the extent of downregulation ofMTHFR activity in response to an inhibitor can be measured in a numberof ways. For example, by measuring the cellular level of mRNA orprotein, by directly measuring the activity of the MTHFR protein, or bymeasuring increases in cellular homocysteine, or decreases in cellular5-methyltetrahydrofolate or methionine, following treatment with acandidate inhibitor and comparing the results to those obtained in theabsence of the candidate inhibitor.

[0112] Typically, an effective inhibitor will lower the level of MTHFRmRNA, protein, or enzymatic activity, or the level of5-methyltetrahydrofolate or methionine in cells that have beenadministered with an MTHFR inhibitor by at least 20% when compared tothe corresponding level in the absence of the inhibitor. More typically,the level will be lowered by at least 40%, frequently by at least 60%,by 80%, or by 90% and occasionally by at least 95%. When the level ofcellular homocysteine is measured as an indicator of the effectivenessof the inhibitor, this level is typically at least 20% greater than inthe absence of the inhibitor. More typically, the level will beincreased by at least 40%, frequently by at least 60%, by 80%, or by90%. The present invention also provides for inhibitors that result inlevels of cellular homocysteine as much as 100%, 200% or even 500%greater than in the absence of the inhibitor.

[0113] Alternatively, the level of MTHFR mRNA, protein, or enzymaticactivity in the presence of an inhibitor can be compared to the level incontrol cells that do not express functional MTHFR, such as cellshomozygous for an MTHFR nonsense mutation. In this case, the level istypically equal to or less than 20-fold, more typically 5-fold andfrequently 2-fold over the level in the control cell.

[0114] Testing the Effect of MTHFR Inhibitors on Cancer Cells

[0115] The ability of the inhibitors of the present invention toselectively inhibit the growth of cancer cells can be determined bytreating a suitable cancer cell-line with the inhibitor and comparingthe growth and/or survival of cells thus treated with an appropriatecontrol. In order to determine the selectivity of the inhibitors, anuntransformed cell-line can be treated with the inhibitor and monitoredfor growth and/or survival in a similar manner.

[0116] Examples of suitable cancer cell-lines for testing the effects ofthe MTHFR inhibitors of the present invention include, but are notlimited to, colon carcinoma cell-lines SW40, LOVO, CaCo-2, Colo 320,SW620 and SW1222; neuroblastoma cell-lines BE(2)C and SK-N-F1; breastcancer cell-lines MCF7 and SKBr3; and glioma cell-line U87-lacZ. Manyother appropriate cancer cell-lines are commercially available.Appropriate controls for these tests include untreated cells, cellstreated with a control compound, such as a non-specific inhibitor, oruntransformed cells treated with the inhibitor.

[0117] Typically, the percent of cancerous cells surviving the treatmentis at least 20% lower than the initial number of cancerous cells, asmeasured using any standard assay, such as those described herein. Moretypically, the number is at least 40% lower, often at least 60% lower or80% lower, and occasionally 100% lower. The MTHFR inhibitor of thepresent invention does not significantly affect non-cancerous cells thatare not rapidly proliferating. In one embodiment of the presentinvention the ratio of percent survival of cancer cells over percentsurvival of normal cells, where the normal cells are not rapidlyproliferating, is less than 1. More typically, this ratio is less than0.9, 0.8, 0.7 or 0.6.

[0118] In accordance with the present invention, when the inhibitor isan antisense oligonucleotide, the number of cancerous cells presentafter administration of an MTHFR antisense nucleotide is at least 2-foldlower than the number of cancerous cells present after administration ofa control oligonucleotide that has a polynucleotide sequence less than70% identical to the reverse complement of a region of an MTHFR nucleicacid. More typically, the number is at least 5-fold greater, frequentlyat least 10-fold greater, 20-fold greater and occasionally 50-foldgreater.

[0119] In one embodiment of the present invention, the effect of anantisense oligonucleotide inhibitor is determined by transfecting cancercells with an inhibitor antisense oligonucleotide or a controloligonucleotide. The initial number of cells is determined, for exampleusing a hemocytometer, and the number of cells surviving treatment isdetermined, for example using a colorimetric cell protein assay. Thepercentage of cells surviving the treatment can then be calculated. In arelated embodiment, the specificity of the antisense oligonucleotideinhibitor in decreasing the growth of cancer cells only is measured bytransfecting a fibroblast (i.e. untransformed) cell-line and determiningcell survival as described above.

[0120] Administration of the MTHFR Inhibitors

[0121] The inhibitors of the present invention may be administeredalone, or in the form of a pharmaceutical composition. The presentinvention, therefore, provides pharmaceutical compositions comprisingone or more MTHFR inhibitors and a pharmaceutically acceptable diluentor excipient. In the case of the pharmaceutical compositions thatcomprise an inhibitor according to the present invention that is anantisense oligonucleotide, the antisense oligonucleotide may be presentas a vector encoding the antisense oligonucleotide. Similarly, in thecase where the pharmaceutical composition comprises an inhibitoraccording to the present invention that is a proteinaceous molecule(i.e. an MTHFR fragment, an MTHFR mutant, an MTHFR specific antibody ora fragment thereof) the molecule may be present as a nucleic acid thatencodes the molecule. In a related embodiment this nucleic acid ispresent in a vector.

[0122] The inhibitors of the present invention and pharmaceuticalcompositions comprising the inhibitors may be administered in a numberof ways depending upon whether local or systemic treatment is desiredand upon the area to be treated. Administration may be topical(including ophthalmic and to mucous membranes including vaginal andrectal delivery), pulmonary, e.g. by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection or infusion; or intracranial,e.g. intrathecal or intraventricular, administration.

[0123] The inhibitors of the present invention may be delivered alone orin combination, and may be delivered along with a pharmaceuticallyacceptable vehicle. Ideally, such a vehicle would enhance the stabilityand/or delivery properties. The present invention also provides foradministration of the inhibitors or pharmaceutical compositionscomprising the inhibitors using a suitable vehicle, such as a liposome,microparticle or microcapsule. In various embodiments of the invention,the use of such vehicles may be beneficial in achieving sustainedrelease of the active component.

[0124] For administration to an individual for the treatment of cancer,the present invention also contemplates the formulation of theinhibitors or pharmaceutical compositions comprising the inhibitors intooral dosage forms such as tablets, capsules and the like. For thispurpose, the inhibitors or pharmaceutical compositions comprising theinhibitors can be combined with conventional carriers, such as magnesiumcarbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin,starch, gelatin, tragacanth, methylcellulose, sodiumcarboxymethyl-cellulose, low melting wax, cocoa butter and the like.Diluents, flavoring agents, solubilizers, lubricants, suspending agents,binders, tablet-disintegrating agents and the like can also be employed,if required. The inhibitors or pharmaceutical compositions comprisingthe inhibitors can be encapsulated with or without other carriers. Inall cases, the proportion of active ingredients in any solid and liquidcomposition will be at least sufficient to impart the desired activityto the individual being treated upon oral administration. The presentinvention further contemplates parenteral injection of the inhibitors orpharmaceutical compositions comprising the inhibitors, in which casethey are used in the form of a sterile solution containing othersolutes, for example, enough saline or glucose to make the solutionisotonic.

[0125] For administration by inhalation or insufflation, the inhibitorsor pharmaceutical compositions comprising the inhibitors can beformulated into an aqueous or partially aqueous solution, which can thenbe utilized in the form of an aerosol. The present invention alsocontemplates topical use of the inhibitors or pharmaceuticalcompositions comprising the inhibitors. For this purpose they can beformulated as dusting powders, creams or lotions in pharmaceuticallyacceptable vehicles, which are applied to affected portions of the skin.

[0126] The present invention also provides for administration ofantisense oligonucleotide, protein and peptide inhibitors in the form ofa genetic vector construct that is designed to direct the in vivosynthesis of the inhibitor. Suitable vectors include viral vectors, suchas an adenoviral, adeno-associated viral, retroviral, lentiviral,baculovirus, or herpes viral vectors. Within the vector construct, thenucleic acid sequence encoding the inhibitor is under the control of asuitable promoter. As described herein, the vector construct mayadditionally contain other regulatory control elements to provideefficient transcription and/or translation of the nucleic acid encodingthe inhibitor.

[0127] The preparation of a vector comprising a nucleic acid sequenceencoding and antisense oligonucleotide according to the presentinvention has been described herein. A worker skilled in the art wouldreadily appreciate that a vector comprising the coding sequence for aproteinaceous inhibitor according to the present invention can beprepare using the same standard techniques.

[0128] Methods of constructing and administering such genetic vectorconstructs for the in vivo synthesis of antisense oligonucleotides,proteins or peptides are well-known in the art. For example, seeAusubel, et al., (2000) Current Protocols in Molecular Biology, Wiley &Sons, New York, N.Y. An efficient method for the introduction,expression and accumulation of antisense oligonucleotides in the cellnucleus is described in U.S. Pat. No. 6,265,167. This method allows theantisense oligonucleotide to hybridize to the sense mRNA in the nucleus,and thereby prevents the antisense oligonucleotide being eitherprocessed or transported into the cytoplasm.

[0129] The dosage requirements for the inhibitors of the presentinvention or pharmaceutical compositions comprising the inhibitors varywith the particular compositions employed, the route of administration,the severity of the symptoms presented and the particular subject beingtreated. Dosage requirements can be determined by standard clinicaltechniques known to a worker skilled in the art. Treatment willgenerally be initiated with small dosages less than the optimum dose ofthe compound. Thereafter the dosage is increased until the optimumeffect under the circumstances is reached. In general, the inhibitors orpharmaceutical compositions comprising the inhibitors are administeredat a concentration that will generally afford effective results withoutcausing any harmful or deleterious side effects. Administration can beeither as a single unit dose or, if desired, the dosage can be dividedinto convenient subunits that are administered at suitable timesthroughout the day.

[0130] Applications

[0131] The present invention provides MTHFR inhibitors that selectivelydecrease the growth of cancer cells, while leaving non-cancerous cellsfully or partly unaffected. The inhibitors of the present invention,therefore, can be used to treat, stabilize or prevent cancer. In thiscontext, the inhibitors exert cytotoxic or cytostatic effects that causea reduction in the size of a tumor, slow or prevent an increase in thesize of a tumor, increase the disease-free survival time between thedisappearance of a tumor and its reappearance, prevent an initial orsubsequent occurrence of a tumor (e.g. metastasis), or reduce an adversesymptom associated with a tumor.

[0132] The present invention also contemplates the use of the MTHFRinhibitors as “sensitizing agents,” which selectively inhibit the growthof cancer cells. In this case, the inhibitor alone does not have acytotoxic effect on the cell, but selectively arrests or slows thegrowth of cancer cells. The inhibitor thus provides a means of weakeningthe cancer cells, and thereby facilitates the benefit from conventionalanti-cancer therapeutics.

[0133] In one embodiment of the present invention one or more MTHFRinhibitor is administered with one or more anti-cancer therapeutics. Theone or more MTHFR inhibitor is administered before during or aftertreatment with the anti-cancer therapeutic. An “anti-cancer therapeutic”is any compound, composition or treatment that prevents or delays thegrowth and/or metastasis of cancer cells. Such anti-cancer therapeuticsinclude but are not limited to chemotherapeutic drug treatment,radiation, gene therapy, hormonal manipulation, immunotherapy andantisense oligonucleotide therapy. It is to be understood thatanticancer therapeutics for use in the present invention also includenovel compounds or treatments developed in the future.

[0134] In accordance with the present invention the one or more MTHFRinhibitors or pharmaceutical compositions comprising the one or moreMTHFR inhibitors is used to selectively inhibit cancer cells in vitro orin vivo, while leaving normal cells fully or partially unaffected. Afurther embodiment of the present invention provides a method fortreating a mammal suffering from cancer by administering one or moreMTHFR inhibitors or a pharmaceutical composition comprising one or moreMTHFR inhibitors. In a related embodiment the MTHFR inhibitor orpharmaceutical compositions is used to selectively inhibit the growthand/or metastasis of cancer cells in vitro or in vivo in a mammal inneed of such therapy. In a specific embodiment of the present inventionthe mammal is a human.

[0135] Examples of cancers that can be treated, stabilized, or preventedin accordance with the present invention include, but are not limitedto, breast carcinomas, colon carcinomas, colorectal carcinomas,neuroblastomas, and gliomas. As used herein, “cancer” refers to alltypes of cancer or neoplasm or malignant tumors found in mammals,including carcinomas and sarcomas. Examples of cancers are cancer of thebrain, breast, cervix, colon, head and neck, kidney, lung, non-smallcell lung, melanoma, mesothelioma, ovary, sarcoma, stomach, uterus andMedulloblastoma.

[0136] The term “leukemia” refers broadly to progressive, malignantdiseases of the blood-forming organs and is generally characterized by adistorted proliferation and development of leukocytes and theirprecursors in the blood and bone marrow. Leukemia is generallyclinically classified on the basis of (1) the duration and character ofthe disease—acute or chronic; (2) the type of cell involved; myeloid(myelogenous), lymphoid (lymphogenous), or monocytic; and (3) theincrease or non-increase in the number of abnormal cells in theblood—leukemic or aleukemic (subleukemic). Leukemia includes, forexample, acute nonlymphocytic leukemia, chronic lymphocytic leukemia,acute granulocytic leukemia, chronic granulocytic leukemia, acutepromyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, aleukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovineleukemia, chronic myelocytic leukemia, leukemia cutis, embryonalleukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia,hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia,stem cell leukemia, acute monocytic leukemia, leukopenic leukemia,lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia,lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia,mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia,monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloidgranulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasmacell leukemia, plasmacytic leukemia, promyelocytic leukemia, Rieder cellleukemia, Schilling's leukemia, stem cell leukemia, subleukemicleukemia, and undifferentiated cell leukemia.

[0137] The term “sarcoma” generally refers to a tumor which is made upof a substance like the embryonic connective tissue and is generallycomposed of closely packed cells embedded in a fibrillar or homogeneoussubstance. Sarcomas include chondrosarcoma, fibrosarcoma, lymphosarcoma,melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, adiposesarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma,botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma,Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing'ssarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma,granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmentedhemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma,immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma,Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymomasarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma,serocystic sarcoma, synovial sarcoma, and telangiectaltic sarcoma.

[0138] The term “melanoma” is taken to mean a tumor arising from themelanocytic system of the skin and other organs. Melanomas include, forexample, acral-lentiginous melanoma, amelanotic melanoma, benignjuvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passeymelanoma, juvenile melanoma, lentigo maligna melanoma, malignantmelanoma, nodular melanoma, subungal melanoma, and superficial spreadingmelanoma.

[0139] The term “carcinoma” refers to a malignant new growth made up ofepithelial cells tending to infiltrate the surrounding tissues and giverise to metastases. Exemplary carcinomas include, for example, acinarcarcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cysticcarcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolarcarcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinomabasocellulare, basaloid carcinoma, basosquamous cell carcinoma,bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogeniccarcinoma, cerebriforn carcinoma, cholangiocellular carcinoma, chorioniccarcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma,cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum,cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma,carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiermoidcarcinoma, carcinoma epitheliale adenoides, exophytic carcinoma,carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma,gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare,glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma,hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma,hyaline carcinoma, hypemephroid carcinoma, infantile embryonalcarcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelialcarcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cellcarcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatouscarcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullarycarcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma,carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma,carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes,naspharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans,osteoid carcinoma, papillary carcinoma, periportal carcinoma,preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma,renal cell carcinoma of kidney, reserve cell carcinoma, carcinomasarcomatodes, schneiderian carcinoma, scirrhous carcinoma, carcinomascroti, signet-ring cell carcinoma, carcinoma simplex, small-cellcarcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cellcarcinoma, carcinoma spongiosum, squamous carcinoma, squamous cellcarcinoma, string carcinoma, carcinoma telangiectaticum, carcinomatelangiectodes, transitional cell carcinoma, carcinoma tuberosum,tuberous carcinoma, verrucous carcinoma, and carcinoma villosum.

[0140] Additional cancers include, for example, Hodgkin's Disease,Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast cancer,ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis,primary macroglobulinemia, small-cell lung tumors, primary brain tumors,stomach cancer, colon cancer, malignant pancreatic insulanoma, malignantcarcinoid, urinary bladder cancer, premalignant skin lesions, testicularcancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer,genitourinary tract cancer, malignant hypercalcemia, cervical cancer,endometrial cancer, adrenal cortical cancer, and prostate cancer.

[0141] Kits

[0142] The present invention additionally provides for therapeutic kitscontaining the inhibitors in pharmaceutical compositions for use in thetreatment of cancer. The contents of the kit can be lyophilized and thekit can additionally contain a suitable solvent for reconstitution ofthe lyophilized components. Individual components of the kit would bepackaged in separate containers and, associated with such containers,can be a notice in the form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals or biologicalproducts, which notice reflects approval by the agency of manufacture,use or sale for human administration.

[0143] When the components of the kit are provided in one or more liquidsolutions, the liquid solution can be an aqueous solution, for example asterile aqueous solution. For in vivo use, the expression construct maybe formulated into a pharmaceutically acceptable syringeablecomposition. In this case the container means may itself be an inhalant,syringe, pipette, eye dropper, or other such like apparatus, from whichthe formulation may be applied to an infected area of the animal, suchas the lungs, injected into an animal, or even applied to and mixed withthe other components of the kit.

[0144] The components of the kit may also be provided in dried orlyophilized forms. When reagents or components are provided as a driedform, reconstitution generally is by the addition of a suitable solvent.It is envisioned that the solvent also may be provided in anothercontainer means. Irrespective of the number or type of containers, thekits of the invention also may comprise, or be packaged with, aninstrument for assisting with the injection/administration or placementof the ultimate complex composition within the body of an animal. Suchan instrument may be an inhalant, syringe, pipette, forceps, measuredspoon, eye dropper or any such medically approved delivery vehicle.

[0145] To gain a better understanding of the invention described herein,the following examples are set forth. It should be understood that theseexamples are for illustrative purposes only. Therefore, they should notlimit the scope of this invention in any way.

EXAMPLES

[0146] General

[0147] Cell Lines

[0148] Human fibroblasts MCH 51, MCH 75 and WG 1554 were obtained fromthe Repository for Mutant Human Cell Strains (Montreal Children'sHospital, Montreal, Canada). WG 1554 is homozygous for a nonsensemutation in MTHFR (Goyette et al, (1994) Nature Genetics, 7:175-200).Human colon carcinoma cell lines CaCo-2, Colo 320, and SW620 wereobtained from American Type Culture Collection (Rockville Md.); humancolon carcinoma cell line SW 1222 was a gift from Dr. N. Beauchemin(McGill University, Montreal, Canada). The carcinoma lines weregenotyped for the MTHFR variant at bp 677 by PCR amplification and Hinfldigestion, as previously reported (Frosst et al, (1995) Nature Genetics,10:111-113). SW 1222 and CaCo-2 were shown to carry the wild typealanine allele (A) whereas Colo 320 and SW620 carry the mutant valineallele (V). Two neuroblastoma lines, BE(2)C and SK-N-F1, were obtainedfrom the American Type Culture Collection. The breast carcinoma celllines, MCF7 and SKBr3, were a gift from Dr. Morag Park (McGillUniversity, Montreal, Canada).

[0149] The MCF7 cell line was grown in α-MEM (Life Technologies,Rockville Md.) and the SKBr3 cell line was maintained in D-MEM (LifeTechnologies). Media for both lines was supplemented with 10% fetalbovine serum (Intergen, Purchase N.Y.). All other cell lines were grownin MEM (Life Technologies) supplemented with 5% fetal bovine serum(Intergen) and 5% iron enriched calf serum (Intergen).

[0150] All media was also supplemented with 50 IU/ml penicillin (LifeTechnologies), 50 μg/ml streptomycin (Life Technologies), 0.5 μg/mlfungizone reagent (Life Technologies). All cell lines were cultured in75 cm² flasks in a humidified 37° C. incubator in 5% CO₂.

[0151] Deficient Culture Media

[0152] MEM and MEM without folate and methionine supplemented with 100mM sodium pyruvate (F−M−) were obtained from Life Technologies. Formethionine-deficient (M−) media, 2.3 μM folate (Sigma-Aldrich, OakvilleON) was added to the F−M− media. For all media, 5% fetal bovine serum(Intergen), 5% iron enriched calf serum (Intergen), 50 IU/ml penicillin(Life Technologies), 50 μg/ml streptomycin (Life Technologies), and 0.5μg/ml fungizone reagent (Life Technologies) were added. For M−H+ media,0.44 mM homocysteine (Sigma-Aldrich) and 1.5 μM vitamin B₁₂(Sigma-Aldrich) was added to the M− media. Dialyzed serum was used forall deficient media.

[0153] Cell Survival Studies of Cells in Deficient Media

[0154] Cell viability studies were performed in 6-well tissue cultureplates starting with 30,000-50,000 cells per well and 3 replicates foreach condition. The initial number of cells were estimated with ahemacytometer. Cell survival in MEM was used as a control forproliferation in deficient media (M−, M−H+). Surviving cells werecounted using the FluoroReporter Colorimetric Cell Protein Assay Kit(Molecular Probes, Eugene Oreg.).

[0155] Oligonucleotides

[0156] For assays to determine the effect of an MTHFR antisenseoligonucleotide on cell viability, an antisense oligonucleotide, EX5,(phosphorothioate 5′-AGCTGCCGAAGGGAGTGGTA-3′) [SEQ ID NO:16] designed tobind exon 5 of MTHFR, a control oligonucleotide with six base pairmismatches, CT 677, (phosphorothioate 5′-TGCTGTCGGAGCGATAGGTC-3′) [SEQID NO:18], and a control oligonucleotide with a scrambled sequence,CTSEX5, (phosphorothioate 5′-GTGACGTAGGACAGCGATGG-3′) [SEQ ID NO:17]were synthesized using standard solid-phase DNA synthesis procedures.This region of exon 5 was chosen because a BLAST search of the humanexpressed sequence (EST) database indicated that this sequence did nothave significant identity to any other reported EST in humans. Inaddition, no sequence variations have been reported for this MTHFR exon,suggesting that an antisense oligonucleotide to exon 5 may bind allMTHFR alleles. The sequences of CT677 and CTSEX5 did not show homologyto any known human genes in a BLAST search. These oligonucleotides weresynthesized as phosphorothioate oligonucleotides in which one of thenon-bridging phosphoryl oxygens of each nucleotide was replaced withsulphur. This modification dramatically improves nuclease stability andpharmacokinetics in vitro and in vivo.

[0157] Transfection with Oligonucleotides and Cell Counting

[0158] Cells were plated in 6-well dishes at 50-70% confluence andincubated overnight in complete medium (Life Technologies). Each wellwas washed once with OPTI-MEM I (Life Technologies). The cells were thenoverlayed with 1 ml of Opti-MEM I media containing 12 μg/ml Lipofectinreagent (Life Technologies) per 400 nM of oligonucleotide. The media wasreplaced with complete media (2-4 ml) after 5 hour incubation at 37° C.with the ASOs. Transfection with oligonucleotides was performed on 3consecutive days followed by a 3-day period of regrowth in MEM. Cellswere counted by SRB staining as outlined in the FluoroReporterColorimetric Cell Protein Assay Kit (Molecular Probes). In eachexperiment, treatments were performed in triplicate.

[0159] In dose response experiments, the total oligonucleotideconcentration was held constant at 400 nM by supplementing the testedoligonucleotide with the control oligonucleotide (Basilion et al, (1999)Molec. Pharmacol., 56:359-369).

[0160] Protein Extraction after Treatment with Oligonucleotides

[0161] For Western blot analysis and MTHFR enzyme assays, 6×10⁵ SW620colon carcinoma cells were plated in 100 mm tissue culture treated petridishes. After transfection of cells with oligonucleotides, the cellswere harvested, and crude protein extracts from cell pellets wereobtained by freezing the pellet at −70° C. and thawing to 4° C. threesuccessive times. The cell pellet was then resuspended in 0.1 M KPO4 pH6.3 with 2 ug/ml aprotinin (Boehringer Mannheim, Laval, Quebec) and 2ug/ml leupeptin (Amersham Pharmacia Biotech, Piscataway N.J.). Cellulardebris was cleared by centrifugation at 14,000 rpm for 10 min. Proteinconcentration was assayed using the Bradford method (Bradford, 1976)according to the manufacturer's instructions (BioRad, Mississauga ON).

[0162] MTHFR Enzyme Assay

[0163] Enzyme activity was measured in the reverse direction, in crudeprotein extracts, as previously described (Christensen et al, (1997)ARTERIOSCLER. Thromb. Vasc. Biol., 17:569-573). Equal amounts of protein(˜60 μg) were used per assay. Enzyme activity was expressed as nmolformaldehyde formed per mg protein/h.

[0164] Western Blot Analysis

[0165] Equal amounts of protein (35-60 μg) were loaded onto a 10% SDSpolyacrylamide gel. Transfer was performed in a transfer buffer (39 mMglycine, 49 mM Tris base, 0.037% SDS, 20% methanol) for 2-3 hour at 70 Vto nitrocellulose (Hybond ECL membrane, Amersham Pharmacia Biotech). Themembrane was blocked with 5% non-fat skim milk in PBS-0.5% Tween 20(Tween 20; BioRad) overnight at 4° C. The MTHFR protein was detectedusing a rabbit anti-porcine MTHFR antibody at a dilution of 1:1000 in 5%non-fat skim milk in PBS-0.5% Tween 20 incubated for 4 to 6 hour at 4°C. After three successive washes in PBS-0.5% Tween 20, anti-rabbithorseradish peroxidase-conjugated antibody (Amersham Pharmacia Biotech)was used as a secondary antibody. The immunocomplexes were visualized byenhanced chemiluminescence with an ECL kit (Amersham Pharmacia Biotech).Quantitation of protein was determined by scanning the films with aflat-bed scanner (Hewlett Packard Scan). The MTHFR and actin band areaswere calculated; MTHFR protein level is expressed as a ratio ofMTHFR/actin.

[0166] Statistical Analysis

[0167] One-way ANOVA was performed using SPSS software, Version 10.0(SPSS Inc., Chicago Ill.), to analyze cell survival after treatment withEX5. The Student t-test was used to evaluate differences in MTHFRactivity, and to analyze cell survival data of fibroblast cell linestreated with EX5 antisense.

Example 1

[0168] Decreased Cell Viability in Cancer Cells after Transfection of anMTHFR Antisense Oligonucleotide

[0169] The following cancer cell lines were used in these experiments:SW620 colon carcinoma (ATCC Accession No. CCL-227), LOVO colon carcinoma(ATCC Accession No. CCL 229), BEC 2 neuroblastoma (ATCC Accession No.CRL 2268), SK-N-F1 neuroblastoma (ATCC Accession No. CRL 2142), MCF7breast cancer (ATCC Accession No. HTB 2), SKBr3 breast cancer (ATCCAccession No. HTB 30), and U87-lacZ glioma cell lines (Li et al., (1999)Clin. Cancer Res. 5:637-642).

[0170] Prior to the transfection of tumor cells with thesephosphorothioate oligonucleotides, the cells were plated at 50-60%confluence in 6-well plates or 10 cm tissue culture dishes and incubatedovernight at 37° C. and 5% CO₂. The next day, the cells were washed withOPTI-MEM I Reduced Serum Media (Gibco, BRL) and treated with theindicated concentration of the MTHR antisense oligonucleotide or one ofthe control oligonucleotides (FIGS. 3-11) and 12 ug/ml LipofectinReagent (Gibco-BRL) in OPTI-MEM I Reduced Serum Media. For the U87-lacZglioma cell line, the control oligonucleotide CT677 was used as thecontrol oligonucleotide; for all the other cell lines, the scrambledoligonucleotide CTSEX5 was used as the control oligonucleotide.

[0171] After a five hour incubation at 37° C. and 5% CO₂ to allowtransfection of the oligonucleotide into the cells, the OPTI-MEM IReduced Serum Media containing the oligonucleotide and Lipofectin wasreplaced with MEM supplemented with 5% FBS and 5% calf serum. The nextday the transfection protocol was repeated. For the second transfection,the cells were washed with Opti-MEM I media and incubated for five hourswith the MTHFR antisense or control oligonucleotide and Lipofectinreagent, as described above. Then, the media was replaced with MEMsupplemented with 5% FBS and 5% calf serum. The following day, a thirdtransfection was performed as described for the second transfection.After this transfection, the cells were allowed to grow in thesupplemented MEM media for two to four days. Then, the number of cellsattached to the tissue culture dish was determined using a colorimetriccell protein assay kit, according to the manufacturer's protocol(FluoroReporter® Colorimetric Cell Protein Assay Kit F-2961 fromMolecular Probes, Eugene Oreg.). This kit contains the anionic xanthenedye, sulforhadamine B, that forms an electrostatically stabilizedcomplex with basic amino acid residues under moderately acidicconditions. This protein-dye complex was detected spectrophotometricallyafter removal of unbound die from TCA-fixed cells.

Example 2

[0172] Failure of an MTHFR Antisense Oligonucleotide to Reduce CellViability of Non-cancerous Cells

[0173] To determine the effect of an MTHFR antisense oligonucleotide onnon-cancerous cells, the human diploid fibroblast cell line WG 1554,which carries 2 nonsense mutations for MTHFR and thus does not producefunctional MTHFR protein, was also tested in the above transfectionassay (Goyette et al., (1994) Nat. Genet. 7:195-200). In particular, thecells were subjected to three rounds of transfection with 400 nM of theantisense oligonucleotide designed to bind exon 5 of MTHFR or thecontrol oligonucleotide and allowed to recover for three days. Incontrast to the previous results with tumor cell lines, there was nodifference in cell survival between WG1554 cells treated with the MTHFRantisense or control oligonucleotide (FIG. 10). This result indicatesthat the MTHFR antisense oligonucleotide is more toxic to cancerouscells than non-cancerous cells that do not express MTHFR. Thus, MTHFRantisense oligonucleotides may produce few adverse side-effects ifadministered to human subjects.

Example 3

[0174] MTHFR Antisense Oligonucleotides Decrease the Cell Viability ofMethionine-dependent Transformed Cells

[0175] Three fibroblast strains (FIG. 11) and 4 colon carcinoma lines(FIG. 12) were grown in MEM, MEM without methionine (M−), or MEM withoutmethionine supplemented with homocysteine and vitamin B₁₂ (M−H+). Thelatter medium served to examine de novo synthesis of methionine fromhomocysteine and 5-methyltetrahydrofolate, catalyzed by vitaminB₁₂-dependent methionine synthase. 5-Methyltetrahydrofolate is theproduct of the MTHFR reaction. All seven lines showed sensitivity to theM− medium; growth was significantly reduced in this medium compared tothat in MEM. Control fibroblasts (MCH 51, MCH 75) could maintainvirtually normal growth in the M−H+ medium. However, the fibroblaststrain WG 1554, which is homozygous for a nonsense mutation in MTHFR(Goyette et al, 1994), was unable to restore growth in the M−H+ medium.The carcinoma lines cultured in the M−H+ medium increased theirproliferation only slightly through endogenous methionine synthesis(FIG. 12). The cell numbers were just a small percentage (5%-20%) of thevalues obtained in MEM. These carcinoma lines are not compromised withrespect to MTHFR activity, although two of the lines (Colo 320 andSW620) have the valine allele, which is associated with mild enzymaticdeficiency.

[0176]FIG. 13A demonstrates a dose-dependent decrease in cell survival(p<0.01, one-way ANOVA) after treatment of SW620 carcinoma cells withthe MTHFR antisense oligonucleotide EX5. At the maximal dose of 400 nM,cell survival decreased approximately 80% compared to that of cellstreated with the scrambled control oligonucleotide CTSEX5.

[0177] To ensure that MTHFR expression was altered, Western blotting wasused to analyze immunoreactive MTHFR protein, after three consecutivetreatments with the EX5 ASO. FIG. 13B demonstrates a significantdecrease in MTHFR protein levels after EX5 treatment, compared totreatment with the scrambled control, CTSEX5, or compared to treatmentwith Lipofectin reagent only (mock transfection). After normalization toactin, MTHFR protein levels following treatment with the control oligowere 94% of mock-treated cells, whereas treatment with 200 nM and 400 nMof EX5, MTHFR protein levels were 39% and 25%, respectively, of that inmock-treated cells (average of 3 Western blots).

Example 4

[0178] Cell Survival of Normal Human Fibroblasts, Breast Carcinoma Cellsand Neuroblastoma Lines After Treatment With 400 nM of EX5.

[0179] After treatment with 400 nM of EX5 as described above, twoneuroblastoma cell lines (BE(2)C and SK-N-F1) showed significantdecreases in cell survival compared to control ASO treated cells:decreases of 80% (p<0.001) and 65% (p<0.01), respectively. Similarly,the breast carcinoma cell line SKBr3 showed a 80% (p<0.0001) decrease incell survival and the MCF7 breast carcinoma line showed a 92% (p<0.0001)reduction in cell survival compared to control oligonucleotide CTSEX5treated cells. Contrary to data obtained in transformed lines, twonormal human fibroblast cell lines (MCH 75 and MCH 51) treated with 400nM of EX5 did not exhibit significant differences in cell survivalcompared to CTSEX5 treated cells (p>0.05). These results are summarizedin FIG. 14.

Example 4

[0180] In vivo Effects of the Downregulation of MTHFR Expression onTumours

[0181] The Min (multiple intestinal neoplasia) mouse is an establishedmouse model for colon cancer. It carries a mutation in the APC gene, thesame gene that is mutated in human hereditary and sporadic colorectaltumours. These mice develop multiple tumors (from 30 to 100) at severalmonths of age.

[0182] Min mice, which carry one copy of the APC mutation, were crossedto MTHFR-deficient mice with a heterozygous knockout of the MTHFR gene.These heterozygous mice carry one copy of the null allele (Mthfr +/−)and, therefore, have 50% of MTHFR activity compared to normal mice.

[0183] Min mice (n=20) carrying just the APC mutation had a mean tumornumber of 75±6.6 (standard error), whereas the Min mice with the APCmutation as well as a MTHFR null allele had a mean tumor number of36±2.7. This difference in tumor number is highly significant(p<0.0001). In addition, the sizes of the tumors were smaller in the Minmice carrying the MTHFR mutation (91.9% of tumors were less than 1 mm),compared to Min mice without the MTHFR mutation (76.3% of tumors wereless than 1 mm).

[0184] Therefore, partial inhibition of MTHFR in transformed cells isassociated with decreased numbers of tumors and decreased tumor growth.Partial inhibition of MTHFR in normal cells does not appear to bedeleterious, since the mice with a heterozygous knockout of MTHFR (Mthfr+/−), without any other mutations, are similar to the normal mice inappearance, birth weight, growth and survival.

[0185] These studies suggest that inhibition of MTHFR may be moredeleterious to rapidly-growing cells than to normal cells. This isconsistent with studies in cultured cells outlined above, whichdemonstrate that transformed cells have a higher requirement formethionine than normal cells.

Example 5

[0186] In Vivo Animal Model for Study of MTHFR AntisenseOligonucleotides

[0187] For in vivo testing of MTHFR inhibitors, the previously describednude mouse cancer model may be used (Bufalo et al., (1996) British J.Canc. 74:387-393; Dean et al., (1996) Cancer Res. 56:3499-3507; Hasegawaet al., (1998) Int. J. Cancer 76:812-816; Narayanan, (1994) In Vivo 8:787-794). Briefly, cancer cells are injected subcutaneously into theflank of a nu/nu athymic mouse. The size of the resulting tumor ismeasured regularly. When the tumor volume reaches 100 mm³-200 mm³, anMTHFR inhibitor or control compound is injected subcutaneously at thesite of the tumor. For initial experiments, a dose of 200 :g inhibitoris injected every other day for a period of one to two weeks. Duringthis period, the appearance and size of the tumor is monitored. Afterthe series of injections is completed, the mouse is sacrificed, and thetumor is removed for further analysis.

[0188] After the efficacy of subcutaneous administration of theinhibitor has been determined, intravenous injections may be performedto evaluate the efficacy of various doses and dosing frequencies forsystemic administration of the inhibitor. This information may be usedto determine the appropriate dosing schedule for human clinical trials.If desired, MTHFR inhibitors may also be tested in other standard animalmodels for cancer, such as naturally-occurring or induced cancers inother mammals including rats, dogs, or monkeys.

[0189] The embodiments of the invention being thus described, it will beobvious that the same may be varied in many ways. Such variations arenot to be regarded as a departure from the spirit and scope of theinvention, and all such modifications as would be obvious to one skilledin the art are intended to be included within the scope of the followingclaims.

1 16 1 2219 DNA Homo sapiens 1 aattccggag ccatggtgaa cgaagccagaggaaacagca gcctcaaccc ctgcttggag 60 ggcagtgcca gcagtggcag tgagagctccaaagatagtt cgagatgttc caccccgggc 120 ctggaccctg agcggcatga gagactccgggagaagatga ggcggcgatt ggaatctggt 180 gacaagtggt tctccctgga attcttccctcctcgaactg ctgagggagc tgtcaatctc 240 atctcaaggt ttgaccggat ggcagcaggtggccccctct acatagacgt gacctggcac 300 ccagcaggtg accctggctc agacaaggagacctcctcca tgatgatcgc cagcaccgcc 360 gtgaactact gtggcctgga gaccatcctgcacatgacct gctgccgtca gcgcctggag 420 gagatcacgg gccatctgca caaagctaagcagctgggcc tgaagaacat catggcgctg 480 cggggagacc caataggtga ccagtgggaagaggaggagg gaggcttcaa ctacgcagtg 540 gacctggtga agcacatccg aagtgagtttggtgactact ttgacatctg tgtggcaggt 600 taccccaaag gccaccccga agcagggagctttgaggctg acctgaagca cttgaaggag 660 aaggtgtctg cgggagccga tttcatcatcacgcagcttt tctttgaggc tgacacattc 720 ttccgctttg tgaaggcatg caccgacatgggcatcactt gccccatcgt ccccgggatc 780 tttcccatcc agggctacca ctcccttcggcagcttgtga agctgtccaa gctggaggtg 840 ccacaggaga tcaaggacgt gattgagccaatcaaagaca acgatgctgc catccgcaac 900 tatggcatcg agctggccgt gagcctgtgccaggagcttc tggccagtgg cttggtgcca 960 ggcctccact tctacaccct caaccgcgagatggctacca cagaggtgct gaagcgcctg 1020 gggatgtgga ctgaggaccc caggcgtcccctaccctggg ctctcagtgc ccaccccaag 1080 cgccgagagg aagatgtacg tcccatcttctgggcctcca gaccaaagag ttacatctac 1140 cgtacccagg agtgggacga gttccctaacggccgctggg gcaattcctc ttcccctgcc 1200 tttggggagc tgaaggacta ctacctcttctacctgaaga gcaagtcccc caaggaggag 1260 ctgctgaaga tgtgggggga ggagctgaccagtgaagcaa gtgtctttga agtctttgtt 1320 ctttacctct cgggagaacc aaaccggaatggtcacaaag tgacttgcct gccctggaac 1380 gatgagcccc tggcggctga gaccagcctgctgaaggagg agctgctgcg ggtgaaccgc 1440 cagggcatcc tcaccatcaa ctcacagcccaacatcaacg ggaagccgtc ctccgacccc 1500 atcgtgggct ggggccccag cgggggctatgtcttccaga aggcctactt agagtttttc 1560 acttcccgcg agacagcgga agcacttctgcaagtgctga agaagtacga gctccgggtt 1620 aattaccacc ttgtcaatgt gaagggtgaaaacatcacca atgcccctga actgcagccg 1680 aatgctgtca cttggggcat cttccctgggcgagagatca tccagcccac cgtagtggat 1740 cccgtcagct tcatgttctg gaaggacgaggcctttgccc tgtggattga gcggtgggga 1800 aagctgtatg aggaggagtc cccgtcccgcaccatcatcc agtacatcca cgacaactac 1860 ttcctggtca acctggtgga caatgacttcccactggaca actgcctctg gcaggtggtg 1920 gaagacacat tggagcttct caacaggcccacccagaatg cgagagaaac ggaggctcca 1980 tgaccctgcg tcctgacgcc ctgcgttggagccactcctg tcccgccttc ctcctccaca 2040 gtgctgcttc tcttgggaac tccactctccttcgtgtctc tcccaccccg gcctccactc 2100 ccccacctga caatggcagc tagactggagtgaggcttcc aggctcttcc tggacctgag 2160 tcggccccac atgggaacct agtactctctgctctaaaaa aaaaaaaaaa aaaggaatt 2219 2 656 PRT Homo sapiens 2 Met ValAsn Glu Ala Arg Gly Asn Ser Ser Leu Asn Pro Cys Leu Glu 1 5 10 15 GlySer Ala Ser Ser Gly Ser Glu Ser Ser Lys Asp Ser Ser Arg Cys 20 25 30 SerThr Pro Gly Leu Asp Pro Glu Arg His Glu Arg Leu Arg Glu Lys 35 40 45 MetArg Arg Arg Leu Glu Ser Gly Asp Lys Trp Phe Ser Leu Glu Phe 50 55 60 PhePro Pro Arg Thr Ala Glu Gly Ala Val Asn Leu Ile Ser Arg Phe 65 70 75 80Asp Arg Met Ala Ala Gly Gly Pro Leu Tyr Ile Asp Val Thr Trp His 85 90 95Pro Ala Gly Asp Pro Gly Ser Asp Lys Glu Thr Ser Ser Met Met Ile 100 105110 Ala Ser Thr Ala Val Asn Tyr Cys Gly Leu Glu Thr Ile Leu His Met 115120 125 Thr Cys Cys Arg Gln Arg Leu Glu Glu Ile Thr Gly His Leu His Lys130 135 140 Ala Lys Gln Leu Gly Leu Lys Asn Ile Met Ala Leu Arg Gly AspPro 145 150 155 160 Ile Gly Asp Gln Trp Glu Glu Glu Glu Gly Gly Phe AsnTyr Ala Val 165 170 175 Asp Leu Val Lys His Ile Arg Ser Glu Phe Gly AspTyr Phe Asp Ile 180 185 190 Cys Val Ala Gly Tyr Pro Lys Gly His Pro GluAla Gly Ser Phe Glu 195 200 205 Ala Asp Leu Lys His Leu Lys Glu Lys ValSer Ala Gly Ala Asp Phe 210 215 220 Ile Ile Thr Gln Leu Phe Phe Glu AlaAsp Thr Phe Phe Arg Phe Val 225 230 235 240 Lys Ala Cys Thr Asp Met GlyIle Thr Cys Pro Ile Val Pro Gly Ile 245 250 255 Phe Pro Ile Gln Gly TyrHis Ser Leu Arg Gln Leu Val Lys Leu Ser 260 265 270 Lys Leu Glu Val ProGln Glu Ile Lys Asp Val Ile Glu Pro Ile Lys 275 280 285 Asp Asn Asp AlaAla Ile Arg Asn Tyr Gly Ile Glu Leu Ala Val Ser 290 295 300 Leu Cys GlnGlu Leu Leu Ala Ser Gly Leu Val Pro Gly Leu His Phe 305 310 315 320 TyrThr Leu Asn Arg Glu Met Ala Thr Thr Glu Val Leu Lys Arg Leu 325 330 335Gly Met Trp Thr Glu Asp Pro Arg Arg Pro Leu Pro Trp Ala Leu Ser 340 345350 Ala His Pro Lys Arg Arg Glu Glu Asp Val Arg Pro Ile Phe Trp Ala 355360 365 Ser Arg Pro Lys Ser Tyr Ile Tyr Arg Thr Gln Glu Trp Asp Glu Phe370 375 380 Pro Asn Gly Arg Trp Gly Asn Ser Ser Ser Pro Ala Phe Gly GluLeu 385 390 395 400 Lys Asp Tyr Tyr Leu Phe Tyr Leu Lys Ser Lys Ser ProLys Glu Glu 405 410 415 Leu Leu Lys Met Trp Gly Glu Glu Leu Thr Ser GluAla Ser Val Phe 420 425 430 Glu Val Phe Val Leu Tyr Leu Ser Gly Glu ProAsn Arg Asn Gly His 435 440 445 Lys Val Thr Cys Leu Pro Trp Asn Asp GluPro Leu Ala Ala Glu Thr 450 455 460 Ser Leu Leu Lys Glu Glu Leu Leu ArgVal Asn Arg Gln Gly Ile Leu 465 470 475 480 Thr Ile Asn Ser Gln Pro AsnIle Asn Gly Lys Pro Ser Ser Asp Pro 485 490 495 Ile Val Gly Trp Gly ProSer Gly Gly Tyr Val Phe Gln Lys Ala Tyr 500 505 510 Leu Glu Phe Phe ThrSer Arg Glu Thr Ala Glu Ala Leu Leu Gln Val 515 520 525 Leu Lys Lys TyrGlu Leu Arg Val Asn Tyr His Leu Val Asn Val Lys 530 535 540 Gly Glu AsnIle Thr Asn Ala Pro Glu Leu Gln Pro Asn Ala Val Thr 545 550 555 560 TrpGly Ile Phe Pro Gly Arg Glu Ile Ile Gln Pro Thr Val Val Asp 565 570 575Pro Val Ser Phe Met Phe Trp Lys Asp Glu Ala Phe Ala Leu Trp Ile 580 585590 Glu Arg Trp Gly Lys Leu Tyr Glu Glu Glu Ser Pro Ser Arg Thr Ile 595600 605 Ile Gln Tyr Ile His Asp Asn Tyr Phe Leu Val Asn Leu Val Asp Asn610 615 620 Asp Phe Pro Leu Asp Asn Cys Leu Trp Gln Val Val Glu Asp ThrLeu 625 630 635 640 Glu Leu Leu Asn Arg Pro Thr Gln Asn Ala Arg Glu ThrGlu Ala Pro 645 650 655 3 346 DNA Homo sapiens misc_feature(326)...(328) n= a, t, g or c 3 gggtgtggct gcctgccccc tgatgctccctgccccaccc tgtgcagtag gaacccagcc 60 atggtgaacg aagccagagg aaacagcagcctcaacccct gcttggaggg cagtgccagc 120 agtggcagtg agagctccaa agatagttcgagatgttcca ccccgggcct ggaccctgag 180 cggcatgaga gactccggga gaagatgaggcggcgattgg aatctggtga caagtggttc 240 tccctggaat tcttccctcc tcgaactgctgagggagctg tcaatctcat ctcaaggtaa 300 actcatgcaa ggttaaggtg ggaggnnngagtggtggtgc ctgggg 346 4 339 DNA Homo sapiens 4 acggatggta tttctcctggaacctctctt cagaaacaaa ccccctacag gtttgaccgg 60 atggcagcag gtggccccctctacatagac gtgacctggc acccagcagg tgaccctggc 120 tcagacaagg agacctcctccatgatgatc gccagcaccg ccgtgaacta ctgtggcctg 180 gagaccatcc tgcacatgacctgctgccgt cagcgcctgg aggagatcac gggccatctg 240 cacaaagcta agcagctgggcctgaagaac atcatggcgc tgcggggagg tgtggagcca 300 gcactcccct acactctgggttctggcttt cccggaggc 339 5 210 DNA Homo sapiens 5 tctggaggtt gggtgagacccagtgactat gacctccacc aaccctgcag acccaatagg 60 tgaccagtgg gaagaggaggagggaggctt caactacgca gtggacctgg tgaagcacat 120 ccgaagtgag tttggtgactactttgacat ctgtgtggca ggtgagtggc tggatcatcc 180 tggtggcggg gatggagctagggaggctga 210 6 294 DNA Homo sapiens misc_feature (260) n = a, t, g orc 6 ccttgaacag gtggaggcca gcctctcctg actgtcatcc ctattggcag gttaccccaa 60aggccacccc gaagcaggga gctttgaggc tgacctgaag cacttgaagg agaaggtgtc 120tgcgggagcc gatttcatca tcacgcagct tttctttgag gctgacacat tcttccgctt 180tgtgaaggca tgcaccgaca tgggcatcac ttgccccatc gtccccggga tctttcccat 240ccaggtgagg ggcccaggan agcccataag ctccctccac cccactctca ccgc 294 7 341DNA Homo sapiens 7 gctggccagc agccgccaca gcccctcatg tcttggacagggctaccact cccttcggca 60 gcttgtgaag ctgtccaagc tggaggtgcc acaggagatcaaggacgtga ttgagccaat 120 caaagacaac gatgctgcca tccgcaacta tggcatcgagctggccgtga gcctgtgcca 180 ggagcttctg gccagtggct tggtgccagg cctccacttctacaccctca accgcgagat 240 ggctaccaca gaggtgctga agcgcctggg gatgtggactgaggacccca ggtgagggca 300 gtggcccaga gatccccaga ggagggtcca agagcagccc c341 8 235 DNA Homo sapiens 8 tccctctagc caatcccttg tctcaattct ctgtccccatcctcacccag gcgtccccta 60 ccctgggctc tcagtgccca ccccaagcgc cgagaggaagatgtacgtcc catcttctgg 120 gcctccagac caaagagtta catctaccgt acccaggagtgggacgagtt ccctaacggc 180 cgctggtgag ggcctgcaga ccttccttgc aaatacatctttgttcttgg gagcg 235 9 281 DNA Homo sapiens 9 actgccctct gtcaggagtgtgccctgacc tctgggcacc cctctgccag gggcaattcc 60 tcttcccctg cctttggggagctgaaggac tactacctct tctacctgaa gagcaagtcc 120 cccaaggagg agctgctgaagatgtggggg gaggagctga ccagtgaagc aagtgtcttt 180 gaagtctttg ttctttacctctcgggagaa ccaaaccgga atggtcacaa agtgagtgat 240 gctggaagtg gggaccctggttcatcccct gcccctggcc t 281 10 283 DNA Homo sapiens 10 cagggtgccaaacctgatgg tcgccccagc cagctcaccg tctctcccag gtgacttgcc 60 tgccctggaacgatgagccc ctggcggctg agaccagcct gctgaaggag gagctgctgc 120 gggtgaaccgccagggcatc ctcaccatca actcacagcc caacatcaac gggaagccgt 180 cctccgaccccatcgtgggc tggggcccca gcgggggcta tgtcttccag aaggtgtggt 240 agggaggcacggggtgcccc cctctcttga ccggcacccg tgg 283 11 202 DNA Homo sapiensmisc_feature (187) n = a, t, g or c 11 gggcgtctgg cagggctggg gttggtgacaggcacctgtc tctcccacag gcctacttag 60 agtttttcac ttcccgcgag acagcggaagcacttctgca agtgctgaag aagtacgagc 120 tccgggttaa ttaccacctt gtcaatgtgaaggtaggcca ggccccacgg ttcccacaga 180 gtaccangcc cttcnttgaa ca 202 12 220DNA Homo sapiens 12 actccagttg ttcttggccc aggtcttacc cccaccccacatcccctcag ggtgaaaaca 60 tcaccaatgc ccctgaactg cagccgaatg ctgtcacttggggcatcttc cctgggcgag 120 agatcatcca gcccaccgta gtggatcccg tcagcttcatgttctggaag gtaaaggagc 180 gggggcaagc ttgccccgcc cacctggaaa accgtgggga220 13 522 DNA Homo sapiens misc_feature (512) n = a, t, g or c 13ctctgtgtgt gtgtgcatgt gtgcgtgtgt gcgggggtat gtgtgtgtag gacgaggcct 60ttgccctgtg gattgagcgg tggggaaagc tgtatgagga ggagtccccg tcccgcacca 120tcatccagta catccacgac aactacttcc tggtcaacct ggtggacaat gacttcccac 180tggacaactg cctctggcag gtggtggaag acacattgga gcttctcaac aggcccaccc 240agaatgcgag agaaacggag gctccatgac cctgcgtcct gacgccctgc gttggagcca 300ctcctgtccc gccttcctcc tccacagtgc tgcttctctt gggaactcca ctctccttcg 360tgtctctccc accccggcct ccactccccc acctgacaat ggcagctaga ctggagtgag 420gcttccaggc tcttcctgga cctgagtcgg ccccacatgg gaacctagta ctctctgctc 480tagccaggag tctgtgctct tttggtgggg ancacttgcn tc 522 14 20 DNA ArtificialSequence Synthetic Primer 14 agctgccgaa gggagtggta 20 15 20 DNAArtificial Sequence Antisense Oligonucleotide 15 gtgacgtagg acagcgatgg20 16 20 DNA Artificial Sequence Antisense Oligonucleotide 16 tgctgtcggagcgataggtc 20

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. An inhibitor ofmethylenetetrahydrofolate reductase (MTHFR) for use in selectiveinhibition of cancer cell growth in a mammal in need of such therapy,wherein the inhibitor reduces MTHFR gene expression or MTHFR activity.2. The inhibitor according to claim 1, which is a non-allele specificantisense oligonucleotide at least 7 nucleotides in length thatcomprises a sequence that is complementary to a gene encoding MTHFR. 3.The inhibitor according to claim 2, wherein the oligonucleotidecomprises a sequence as set forth in SEQ ID NO:16.
 4. The inhibitoraccording to claim 1, which is a ribozyme.
 5. The inhibitor according toclaim 1, which is an antibody or an antibody fragment that binds to andinhibits MTHFR.
 6. The inhibitor according to claim 1, which is a smallmolecule.
 7. The inhibitor according to claim 5, which is a peptide. 8.The inhibitor according to claim 1, which is a mutant MTHFR or afragment of MTHFR.
 9. The inhibitor according to any one of claims 1, 2,3, 4, 5, 6, 7, or 8, wherein said inhibition of cancer cell growthsensitizes the cancer cells to an anti-cancer therapeutic.
 10. Theinhibitor according to any one of claims 1, 2, 3, 4, 5, 6, 7, 8, or 9,wherein said mammal has cancer.
 11. The inhibitor according to claim 10,wherein said cancer is a breast cancer, colon carcinoma, colorectalcarcinoma, neuroblastoma, or glioma.
 12. The inhibitor according to anyone of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, wherein said mammalis a human.
 13. A nucleic acid comprising a sequence encoding theinhibitor according to any one of claims 2, 3, 4, 5, 7 or
 8. 14. Thenucleic acid according to claim 13, which is operatively associated witha regulatory control element.
 15. The nucleic acid according to claim14, wherein said regulatory control element is a transcriptional controlelement.
 16. A vector comprising the nucleic acid according to claim 13,14, or
 15. 17. A pharmaceutical composition comprising the inhibitoraccording to any one of claims 1, 2, 3, 4, 5, 6, 7, or 8, and apharmaceutically acceptable carrier, diluent or excipient.
 18. Use of amethylenetetrahydrofolate reductase (MTHFR) inhibitor for themanufacture of a medicament, wherein the inhibitor reduces MTHFR geneexpression or MTHFR activity.
 19. The use according to claim 18, whereinthe inhibitor is a non-allele specific antisense oligonucleotide atleast 7 nucleotides in length that comprises a sequence that iscomplementary to a gene encoding MTHFR.
 20. The use according to claim19, wherein the oligonucleotide comprises SEQ ID NO:16.
 21. The useaccording to claim 18, wherein the inhibitor is an antibody or anantibody fragment that binds to and inhibits MTHFR.
 22. The useaccording to claim 18, wherein the inhibitor is a small molecule. 23.The use according to claim 18, wherein the inhibitor is a mutant MTHFRor a fragment of MTHFR.
 24. The use according to any one of claims 18,19, 20, 21, 22, or 23, wherein the inhibitor sensitizes the cancer cellsto an anti-cancer therapeutic.
 25. The use according to any one ofclaims 18, 19, 20, 21, 22, or 23, wherein the medicament additionallycomprises an anti-cancer therapeutic.
 26. The use according to any oneof claims 18, 19, 20, 21, 22, 23, 24, or 25, wherein the medicament isfor treating, stabilizing or preventing cancer in a mammal.
 27. The useaccording to claim 26, wherein said cancer is a breast cancer, coloncarcinoma, colorectal carcinoma, neuroblastoma, or glioma.
 28. The useaccording to any one of claims 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27,wherein the mammal is a human.
 29. Use of the vector according to claim16 for the manufacture of a medicament, wherein the inhibitorselectively inhibits cancer cell growth and wherein the inhibitorreduces methylenetetrahydrofolate reductase (MTHFR) gene expression orMTHFR activity.
 30. The use according to claim 29, wherein the inhibitorsensitizes the cancer cells to an anti-cancer therapeutic.
 31. The useaccording to claim 29, wherein the medicament additionally comprises ananti-cancer therapeutic.
 32. The use according to any one of claims 29,30, or 31, wherein the medicament is for treating, stabilizing orpreventing cancer in a mammal.
 33. The use according to claim 32,wherein wherein said cancer is a breast cancer, colon carcinoma,colorectal carcinoma, neuroblastoma, or glioma.
 34. The use according toany one of claims 29, 30, 31, 32, or 33, wherein the mammal is a human.35. A non-allele specific antisense oligonucleotide at least 7nucleotides in length that comprises a sequence that is complementary toa gene encoding human methylenetetrahydrofolate reductase (MTHFR),wherein the oligonucleotide inhibits human MTHFR gene expression. 36.The antisense oligonucleotide according to claim 35, wherein theantisense oligonucleotide selectively inhibits cancer cell growth. 37.The antisense oligonucleotide according to claim 35 or 36, wherein theantisense oligonucleotide is complementary to exon 5 of the MTHFR gene.38. The antisense oligonucleotide according to any one of claims 35, 36,or 37, wherein the oligonucleotide comprises a sequence as set forth inSEQ ID NO:16.
 39. The antisense oligonucleotide according to any one ofclaims 35, 36, 37, or 38, wherein the oligonucleotide is aphosphorothioate nucleic acid.
 40. A vector comprising a nucleic acidencoding the antisense oligonucleotide according to any one of claims35, 36, 37, 38, or
 39. 41. A method of treating stabilizing orpreventing cancer in a mammal comprising the step of selectivelyinhibiting cancer cell growth in the mammal by administering aninhibitor of methylenetetrahydrofolate reductase (MTHFR), wherein theinhibitor reduces MTHER gene expression or MTHFR activity.
 42. Themethod according to claim 41, wherein said mammal is a human.
 43. Amethod of inhibiting growth of cancer cells comprising the step ofcontacting said cancer cells with an inhibitor ofmethylenetetrahydrofolate reductase (MTHFR), wherein the inhibitorreduces MTHFR gene expression or MTHFR activity.
 44. A kit for the useaccording to any one of claims 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, or 34, or the method according to any one ofclaims 41, 42, or 43, comprising an inhibitor ofmethylenetetrahydrofolate reductase (MTHFR) which selectively inhibitscancer cell growth, wherein the inhibitor reduces MTHFR gene expressionor MTHFR activity.